Targeting deregulated Wnt signaling in cancer using stabilized alpha-helices of BCL-9

ABSTRACT

The invention provides structurally-constrained peptides by hydrocarbon stapling of a BCL9 HD2 helix for use as a therapeutic agent. The invention further provides methods and kits for use of the structurally-constrained peptide of the instant invention. The invention is based, at least in part, on the results provided herein demonstrating that hydrocarbon stapled helical peptides display excellent proteolytic, acid, and thermal stability, restore the native helical structure of the peptide, possess superior pharmacokinetic properties compared to the corresponding unmodified peptides, and are highly effective in binding to β-catenin in vitro, in cellulo, and in vivo, disrupting the BCL9/β-catenin interaction, and thereby interfering with deregulated Wnt/β-catenin signaling for therapeutic benefit in a variety of human diseases including human cancer.

This application is a divisional application of U.S. application Ser.No. 14/111,299, filed Dec. 6, 2013, now U.S. Pat. No. 10,703,785, whichis a national stage application filed under 35 U.S.C. § 371 ofInternational Application No. PCT/US2012/033822, filed Apr. 16, 2012,which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 61/475,932, filed Apr. 15, 2011, each ofwhich is incorporated herein by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under grant number R01CA151391 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCHII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 31, 2015 isnamed 48289-521N01US_SL.txt and is 80,031 bytes in size.

BACKGROUND

The canonical Wnt pathway regulates the constitutive level andintracellular localization of β-catenin, a key component of a tightlyregulated receptor-mediated signal transduction network required forboth embryonic development and adult tissue homeostasis. In unstimulatednormal cells, β-catenin binds to adenomatous polyposis coli (APC),glycogen synthase kinase 3β (GSK3β), and Axin, which form a destructioncomplex that phosphorylates β-catenin, targeting it for proteosomaldegradation. The binding of Wnt ligands to the frizzled and low-densitylipoprotein receptors (LRP5 and LRP6) inhibits the activity of theGSK3β/APC/Axin complex, enabling non-phosphorylated β-catenin to undergonuclear translocation to exert its transcriptional effects. Nuclearβ-catenin associates with the lymphoid enhancer factor/T-cell factor(LEF/TCF) family of transcription factors to induce the expression ofcell proliferation, migration, and survival genes, such as c-Myc andcyclin D1. Normally, this transcriptional pathway is turned off when Wntligands uncouple from their receptors. However, a variety of loss offunction mutations in APC and Axin, and activating mutations inβ-catenin itself, enable β-catenin to escape the destruction complex,persist in the nucleus, and drive oncogenic transcription.

In Drosophila melanogaster, the transcriptional activity of β-cateninfurther depends on two co-factors, BCL9 and Pygopus. The formation of aquaternary complex consisting of TCF, β-catenin, BCL9, and Pygopusenhances β-catenin-dependent Wnt transcriptional activity. The humanBCL9 gene was first identified by cloning the t(1;14)(q21;q32)translocation from a patient with precursor B-cell acute lymphoblasticleukemia (ALL). Amplifications of the chromosome 1q21-locus in which theBCL9 gene resides is observed in a broad range of human cancer types andit has been associated with tumor progression, decreased survival andpoor clinical outcome. Most recently, insertional mutagenesis by thePiggyBac transposon has identified a hit in BCL9. Whereas in colorectalcancer (CRC) established mutations in APC and β-catenin drive theoncogenic phenotype, in multiple myeloma (MM) no such mutations havebeen reported and Wnt activation is instead driven by BCL9, implicatingthis β-catenin co-factor as a bona-fide oncogene. BCL9 overexpressionhas since been identified in a large subgroup of human tumors, yet isnot expressed in the normal cellular counterparts from which the tumorsoriginate. BCL9-mediated enhancement of β-catenin's transcriptionalactivity increases cell proliferation, migration, invasion, and themetastatic potential of tumor cells by promoting the loss of anepithelial phenotype and gain of a mesenchyme-like functionality.shRNA-induced downregulation of BCL9 in vivo suppresses the expressionof Wnt targets c-Myc, cyclin D1, CD44, and VEGF, and correspondinglyincreases the survival of xenograft mice with CRC and MM by reducingtumor load, metastasis, and the host angiogenesis response. The strikingBCL9 dependence of these cancers and the expression of BCL9 in ˜30% ofepithelial tumors provides a compelling rationale for targeting theBCL9/β-catenin protein interaction. Importantly, Bcl9-null mice lack anovert disease phenotype, suggesting that pharmacologic blockade of theBCL9/β-catenin complex may be relatively non-toxic.

The Wnt pathway consists of a tightly regulated receptor-mediated signaltransduction system required for both embryonic development and adulttissue homeostasis in vertebrates and invertebrates and involvescanonical and non-canonical Wnt pathways. Several components of thecanonical Wnt signaling cascade have been shown to function as eithertumor suppressor genes (TSG) or as oncogenes in a wide range of commonhuman cancers including colorectal, hepatocellular, breast, endometrialcarcinomas and MM. Furthermore, the canonical Wnt pathway has beenimplicated in the regulation of normal (e.g., wound healing) as well aspathological processes (e.g., diabetes). These observations underscorethe relevance of this pathway to oncogenesis and the need for furtherinvestigation of Wnt signaling components as potential targets forcancer therapy, wound healing, angiogenesis and diabetes.

It is an object of the invention to design and generatehydrocarbon-stapled peptides of the HD2 domain of BCL9 (stapledα-helices of BCL9 or SAH-BCL9) to block Wnt signaling. It is also anobject to demonstrate that direct binding of stabilized α-helicalpeptides to β-catenin prevents β-catenin/BCL9 interaction, Wnttranscriptional activity, and expression of downstream targets. Suchmechanisms would result in a method of treatment of cancer cells withSAH-BCL9 and result in inhibition of tumor cell proliferation,migration, tumor-induced angiogenesis, tumor load, de-differentiation(epithelial-mesenchymal transition [EMT]), and metastasis inWnt/β-catenin-driven cancers.

SUMMARY OF THE INVENTION

The invention provides structurally-constrained, protease-resistant, andcell-permeable BCL9 α-helical peptides, and methods of use of thosepeptides as therapeutic and prophylactic agents. Suchstructurally-constrained peptides display excellent proteolytic, acid,and thermal stability, and possess superior pharmacokinetic propertiescompared to the corresponding unmodified peptides. The peptides of theinvention are stabilized with at least one hydrocarbon staple, but couldinclude two, three or more hydrocarbon staples. The inclusion ofmultiple hydrocarbon staples is particularly suited for alpha helicalpeptides that are 20 or more amino acids in length. The hydrocarbonstaples allow for the amino acid residues on an interacting face to beproperly oriented due to stabilization of the helical structure of theBCL9 HD2 domain.

In one aspect, the invention provides a structurally constrained peptideof an HD2 domain of BCL9 (BCL9-HD2), comprising at least one hydrocarbonstaple or stitch.

In one embodiment, the peptide comprises an interacting face comprisedof amino acids that interact with β-catenin.

In another embodiment, the interacting face comprises about 3 to about20 amino acids.

In certain embodiments, the interacting face comprises 4-15 amino acids.

In various embodiments, the interacting face comprises 40% or greateridentity to a helical face of BCL9 HD2 that binds β-catenin, wherein theinteracting face comprises BCL9 residues Gln-355, His-358, Arg-359,Ser-362, Leu-363, Leu-366, Ile-369, Gln-370, Leu-373, and Phe-374, orconservative substitutions thereof.

In still other embodiments, the interacting face comprises 50% to 90%identity to the helical face of BCL9 HD2 that binds β-catenin, whereinthe interacting face comprises BCL9 residues Gln-355, His-358, Arg-359,Ser-362, Leu-363, Leu-366, Ile-369, Gln-370, Leu-373, and Phe-374, orconservative substitutions thereof.

In another embodiment, the interacting face represents a single face ofan α-helix.

In various embodiments, the single face of a helix comprises one, two,three, or four adjacent stacked columns of amino acids, wherein thestacked columns of amino acids are defined by positions a, d, and g;positions b and e; or positions c and f; in an alpha helix having 3.6amino acids per turn wherein the amino acids are consecutively andserially assigned positions a-g; and positions a and d; positions b ande; or positions c and fin a 3₁₀ helix having 3 amino acids per turnwherein the amino acids are consecutively and serially assignedpositions a-f; or homologues thereof.

In other embodiments, the invention provides a structurally constrainedpeptide consisting of: between about 20% to 100% sequence homology toamino acids 351 to 374 of BCL9 HD2, SEQ ID NO: 1(LSQEQLEHRERSLQTLRDIQRMLF),

wherein the peptide comprises between one and five hydrocarbon staples.

In other embodiments, the invention provides a structurally constrainedpeptide consisting of: between about 50% to 100% sequence homology toamino acids 351 to 374 of BCL9 HD2, SEQ ID NO: 1(LSQEQLEHRERSLQTLRDIQRMLF),

wherein the peptide comprises between one and five hydrocarbon staples.

In various embodiments, the hydrocarbon staple or stitch is between oneor more natural or non-natural amino acids.

In certain embodiments, the hydrocarbon staple or stitch is formed by anolefin metathesis reaction.

In another embodiment, the non-natural amino acids are selected from thefollowing:

In various embodiments, the structurally constrained peptide comprises 1to 5 staples or stitches within the BCL9 HD2 peptide.

In other embodiments, one staple or stitch is located at the followingpositions within the BCL9 HD2 peptide: a) i, i+4; b) i, i+7; and c) i,i+3.

In certain embodiments, another staple or stitch is located at thefollowing positions within the BCL9 HD2 peptide: a) i, i+4; b) i, i+7;and c) i, i+3.

In various embodiments, any other staples or stitches are located at thefollowing positions within the BCL9 HD2 peptide: a) i, i+4; b) i, i+7;and c) i, i+3.

In another embodiment, the invention provides a structurally constrainedpeptide, wherein one hydrocarbon staple is located at the followingexemplary positions within the BCL9 HD2 peptide, and iterated by staplescanning:

BCL9-HD2 domain (SEQ ID NO: 2) 351 LSQEQLEHRERSLQTLRDIQRBLF 374 i, i + 4 single staples: (SEQ ID NO. 8) XSQEXLEHRERSLQTLRDIQRBLF(SEQ ID NO. 9) LXQEQXEHRERSLQTLRDIQRBLF (SEQ ID NO. 10)LSXEQLXHRERSLQTLRDIQRBLF (SEQ ID NO. 11) LSQXQLEXRERSLQTLRDIQRBLF(SEQ ID NO. 12) LSQEXLEHXERSLQTLRDIQRBLF (SEQ ID NO. 13)LSQEQXEHRXRSLQTLRDIQRBLF (SEQ ID NO. 14) LSQEQLXHREXSLQTLRDIQRBLF(SEQ ID NO. 15) LSQEQLEXRERXLQTLRDIQRBLF (SEQ ID NO. 16)LSQEQLEHXERSXQTLRDIQRBLF (SEQ ID NO. 17) LSQEQLEHRXRSLXTLRDIQRBLF(SEQ ID NO. 18) LSQEQLEHREXSLQXLRDIQRBLF (SEQ ID NO. 19)LSQEQLEHRERXLQTXRDIQRBLF (SEQ ID NO. 20) LSQEQLEHRERSXQTLXDIQRBLF(SEQ ID NO. 21) LSQEQLEHRERSLXTLRXIQRBLF (SEQ ID NO. 22)LSQEQLEHRERSLQXLRDXQRBLF (SEQ ID NO. 23) LSQEQLEHRERSLQTXRDIXRBLF(SEQ ID NO. 24) LSQEQLEHRERSLQTLXDIQXBLF (SEQ ID NO. 25)LSQEQLEHRERSLQTLRXIQRXLF (SEQ ID NO. 26) LSQEQLEHRERSLQTLRDXQRBXF(SEQ ID NO. 27) LSQEQLEHRERSLQTLRDIXRBLX i, i + 7 staples:(SEQ ID NO. 28) XSQEQLEXRERSLQTLRDIQRBLF (SEQ ID NO. 29)LXQEQLEHXERSLQTLRDIQRBLF (SEQ ID NO. 30) LSXEQLEHRXRSLQTLRDIQRBLF(SEQ ID NO. 31) LSQXQLEHREXSLQTLRDIQRBLF (SEQ ID NO. 32)LSQEXLEHRERXLQTLRDIQRBLF (SEQ ID NO. 33) LSQEQXEHRERSXQTLRDIQRBLF(SEQ ID NO. 34) LSQEQLXHRERSLXTLRDIQRBLF (SEQ ID NO. 35)LSQEQLEXRERSLQXLRDIQRBLF (SEQ ID NO. 36) LSQEQLEHXERSLQTXRDIQRBLF(SEQ ID NO. 37) LSQEQLEHRXRSLQTLXDIQRBLF (SEQ ID NO. 38)LSQEQLEHREXSLQTLRXIQRBLF (SEQ ID NO. 39) LSQEQLEHRERXLQTLRDXQRBLF(SEQ ID NO. 40) LSQEQLEHRERSXQTLRDIXRBLF (SEQ ID NO. 41)LSQEQLEHRERSLXTLRDIQXBLF (SEQ ID NO. 42) LSQEQLEHRERSLQXLRDIQRXLF(SEQ ID NO. 43) LSQEQLEHRERSLQTXRDIQRBXF (SEQ ID NO. 44)LSQEQLEHRERSLQTLXDIQRBLX i, i + 3 single staples: (SEQ ID NO. 45)XSQXQLEHRERSLQTLRDIQRBLF (SEQ ID NO. 46) LXQEXLEHRERSLQTLRDIQRBLF(SEQ ID NO. 47) LSXEQXEHRERSLQTLRDIQRBLF (SEQ ID NO. 48)LSQEXLEXRERSLQTLRDIQRBLF (SEQ ID NO. 49) LSQEQXEHXERSLQTLRDIQRBLF(SEQ ID NO. 50) LSQEQLXHRXRSLQTLRDIQRBLF (SEQ ID NO. 51)LSQEQLEXREXSLQTLRDIQRBLF (SEQ ID NO. 52) LSQEQLEHXERXLQTLRDIQRBLF(SEQ ID NO. 53) LSQEQLEHRXRSXQTLRDIQRBLF (SEQ ID NO. 54)LSQEQLEHREXSLXTLRDIQRBLF (SEQ ID NO. 55) LSQEQLEHRERXLQXLRDIQRBLF(SEQ ID NO. 56) LSQEQLEHRERSXQTXRDIQRBLF (SEQ ID NO. 57)LSQEQLEHRERSLXTLXDIQRBLF (SEQ ID NO. 58) LSQEQLEHRERSLQXLRXIQRBLF(SEQ ID NO. 59) LSQEQLEHRERSLQTXRDXQRBLF (SEQ ID NO. 60)LSQEQLEHRERSLQTLXDIXRBLF (SEQ ID NO. 61) LSQEQLEHRERSLQTLRXIQXBLF(SEQ ID NO. 62) LSQEQLEHRERSLQTLRDXQRXLF (SEQ ID NO. 63)LSQEQLEHRERSLQTLRDIXRBXF (SEQ ID NO. 64) LSQEQLEHRERSLQTLRDIQXBLX.

In certain embodiments, the invention provides a structurallyconstrained peptide, wherein two hydrocarbon staples are located at thefollowing exemplary positions within the BCL9 HD2 peptide, and iteratedby staple scanning:

BCL9-HD2 domain (SEQ ID NO. 2) 351 LSQEQLEHRERSLQTLRDIQRBLF 374i, i + 3 double staples: (SEQ ID NO. 65) XSQXQLEHRERSLQTLRDIQXBLX(SEQ ID NO. 66) XSQXQLEHRERSLQTLRDIXRBXF (SEQ ID NO. 67)XSQXQLEHRERSLQTLRDXQRBXF i, i + 4 double staples: (SEQ ID NO. 68)XSQEXLEHRERSLQTLRDIXRBLX (SEQ ID NO. 69) XSQEXLEHRERSLQTLRDXQRBXF(SEQ ID NO. 70) XSQEXLEHRERSLQTLXDIQRXLF i, i + 7 double staples:(SEQ ID NO. 71) XSQEQLEXRERSLQTLXDIQRBLX (SEQ ID NO. 72)XSQEQLEXRERSLQTXRDIQRBXF (SEQ ID NO. 73) XSQEQLEXRERSLQXLRDIQRXLF.

In another embodiment, the invention provides a structurally constrainedpeptide, wherein the one or more hydrocarbon staples or stitches islocated at any of the following exemplary positions within the BCL9 HD9domain and iterated by staple scanning:

BCL9-HD2 domain (SEQ ID NO. 2) 351 LSQEQLEHRERSLQTLRDIQRBLF 374Mixed i, i + 4; i, i + 3; and i, i + 7 double staples: (SEQ ID NO. 74)XSQEXLEHRERSLQTLXDIQRBLX (SEQ ID NO. 75) XSQEXLEHRERSLQTXRDIQRBXF(SEQ ID NO. 76) XSQEXLEHRERSLQXLRDIQRXLF (SEQ ID NO. 77)XSQEXLEHRERSLQTLRDIQXBLX (SEQ ID NO. 78) XSQEXLEHRERSLQTLRDIXRBXF(SEQ ID NO. 79) XSQEXLEHRERSLQTLRDXQRXLF (SEQ ID NO. 80)XSQEQLEXRERSLQTLRDIXRBLX (SEQ ID NO. 81) XSQEQLEXRERSLQTLRDXQRBXF(SEQ ID NO. 82) XSQEQLEXRERSLQTLRXIQRXLF (SEQ ID NO. 83)XSQEQLEXRERSLQTLRDIQXBLX (SEQ ID NO. 84) XSQEQLEXRERSLQTLRDIXRBXF(SEQ ID NO. 85) XSQEQLEXRERSLQTLRDXQRXLF (SEQ ID NO. 86)XSQXQLEHRERSLQTLRDIXRBLX (SEQ ID NO. 87) XSQXQLEHRERSLQTLRDXQRBXF(SEQ ID NO. 88) XSQXQLEHRERSLQTLRXIQRXLF (SEQ ID NO. 89)XSQXQLEHRERSLQTLXDIQRBLX (SEQ ID NO. 90) XSQXQLEHRERSLQTXRDIQRBXF(SEQ ID NO. 91) XSQXQLEHRERSLQXLRDIQRXLF Sequential i, i + 4 staples:(SEQ ID NO. 92) XSQEXLEHXERSLQTLRDIQRBLF (SEQ ID NO. 93)LXQEQXEHRXRSLQTLRDIQRBLF (SEQ ID NO. 94) LSXEQLXHREXSLQTLRDIQRBLF(SEQ ID NO. 95) LSQXQLEXRERXLQTLRDIQRBLF (SEQ ID NO. 96)LSQEXLEHXERSXQTLRDIQRBLF (SEQ ID NO. 97) LSQEQXEHRXRSLXTLRDIQRBLF(SEQ ID NO. 98) LSQEQLXEREXSLQXLRDIQRBLF (SEQ ID NO. 99)LSQEQLEXRERXLQTXRDIQRBLF (SEQ ID NO. 100) LSQEQLEHXERSXQTLXDIQRBLF(SEQ ID NO. 101) LSQEQLEHRXRSLXTLRXIQRBLF (SEQ ID NO. 102)LSQEQLEHREXSLQXLRDXQRBLF (SEQ ID NO. 103) LSQEQLEHRERXLQTXRDIXRBLF(SEQ ID NO. 104) LSQEQLEHRERSXQTLXDIQXBLF (SEQ ID NO. 105)LSQEQLEHRERSLXTLRXIQRXLF (SEQ ID NO. 106) LSQEQLEHRERSLQXLRDXQRBXF(SEQ ID NO. 107) LSQEQLEHRERSLQTXRDIXRBLX Sequential i, i + 3 staples:(SEQ ID NO. 108) XSQXQLXHRERSLQTLRDIQRBLF Sequential i, i + 7 staples:(SEQ ID NO. 109) XSQEQLEXRERSLQXLRDIQRBLF Mixed sequential staples:(SEQ ID NO. 110) XSQXQLEXRERSLQTLRDIQRBLF (SEQ ID NO. 111)XSQXQLEHREXSLQTLRDIQRBLF (SEQ ID NO. 112) XSQEXLEXRERSLQTLRDIQRBLF(SEQ ID NO. 113) XSQEXLEHRERXLQTLRDIQRBLF (SEQ ID NO. 114)XSQEQLEXREXSLQTLRDIQRBLF (SEQ ID NO. 115) XSQEQLEXRERXLQTLRDIQRBLF

In various embodiments, the invention provides a structurallyconstrained peptide, further comprising one to four additionalhydrocarbon staples.

In other embodiments, the invention provides a structurally constrainedpeptide, wherein the additional hydrocarbon staples are located at anyof the following exemplary positions within the BCL9 HD2 domain anditerated by staple scanning:

BCL9-HD2 domain (SEQ ID NO. 2) 351 LSQEQLEHRERSLQTLRDIQRBLF 374i, i + 4 single staples: (SEQ ID NO. 8) XSQEXLEHRERSLQTLRDIQRBLF(SEQ ID NO. 9) LXQEQXEHRERSLQTLRDIQRBLF (SEQ ID NO. 10)LSXEQLXERERSLQTLRDIQRBLF (SEQ ID NO. 11) LSQXQLEXRERSLQTLRDIQRBLF(SEQ ID NO. 12) LSQEXLEHXERSLQTLRDIQRBLF (SEQ ID NO. 13)LSQEQXEHRXRSLQTLRDIQRBLF (SEQ ID NO. 14) LSQEQLXHREXSLQTLRDIQRBLF(SEQ ID NO. 15) LSQEQLEXRERXLQTLRDIQRBLF (SEQ ID NO. 16)LSQEQLEHXERSXQTLRDIQRBLF (SEQ ID NO. 17) LSQEQLEHRXRSLXTLRDIQRBLF(SEQ ID NO. 18) LSQEQLEHREXSLQXLRDIQRBLF (SEQ ID NO. 19)LSQEQLEHRERXLQTXRDIQRBLF (SEQ ID NO. 20) LSQEQLEHRERSXQTLXDIQRBLF(SEQ ID NO. 21) LSQEQLEHRERSLXTLRXIQRBLF (SEQ ID NO. 22)LSQEQLEHRERSLQXLRDXQRBLF (SEQ ID NO. 23) LSQEQLEHRERSLQTXRDIXRBLF(SEQ ID NO. 24) LSQEQLEHRERSLQTLXDIQXBLF (SEQ ID NO. 25)LSQEQLEHRERSLQTLRXIQRXLF (SEQ ID NO. 26) LSQEQLEHRERSLQTLRDXQRBXF(SEQ ID NO. 27) LSQEQLEHRERSLQTLRDIXRBLX i, i + 7 staples:(SEQ ID NO. 28) XSQEQLEXRERSLQTLRDIQRBLF (SEQ ID NO. 29)LXQEQLEHXERSLQTLRDIQRBLF (SEQ ID NO. 30) LSXEQLEHRXRSLQTLRDIQRBLF(SEQ ID NO. 31) LSQXQLEHREXSLQTLRDIQRBLF (SEQ ID NO. 32)LSQEXLEHRERXLQTLRDIQRBLF (SEQ ID NO. 33) LSQEQXEHRERSXQTLRDIQRBLF(SEQ ID NO. 34) LSQEQLXHRERSLXTLRDIQRBLF (SEQ ID NO. 35)LSQEQLEXRERSLQXLRDIQRBLF (SEQ ID NO. 36) LSQEQLEHXERSLQTXRDIQRBLF(SEQ ID NO. 37) LSQEQLEHRXRSLQTLXDIQRBLF (SEQ ID NO. 38)LSQEQLEHREXSLQTLRXIQRBLF (SEQ ID NO. 39) LSQEQLEHRERXLQTLRDXQRBLF(SEQ ID NO. 40) LSQEQLEHRERSXQTLRDIXRBLF (SEQ ID NO. 41)LSQEQLEHRERSLXTLRDIQXBLF (SEQ ID NO. 42) LSQEQLEHRERSLQXLRDIQRXLF(SEQ ID NO. 43) LSQEQLEHRERSLQTXRDIQRBXF (SEQ ID NO. 44)LSQEQLEHRERSLQTLXDIQRBLX i, i + 3 single staples: (SEQ ID NO. 45)XSQXQLEHRERSLQTLRDIQRBLF (SEQ ID NO. 46) LXQEXLEHRERSLQTLRDIQRBLF(SEQ ID NO. 47) LSXEQXEHRERSLQTLRDIQRBLF (SEQ ID NO. 48)LSQEXLEXRERSLQTLRDIQRBLF (SEQ ID NO. 49) LSQEQXEHXERSLQTLRDIQRBLF(SEQ ID NO. 50) LSQEQLXHRXRSLQTLRDIQRBLF (SEQ ID NO. 51)LSQEQLEXREXSLQTLRDIQRBLF (SEQ ID NO. 52) LSQEQLEHXERXLQTLRDIQRBLF(SEQ ID NO. 53) LSQEQLEHRXRSXQTLRDIQRBLF (SEQ ID NO. 54)LSQEQLEHREXSLXTLRDIQRBLF (SEQ ID NO. 55) LSQEQLEHRERXLQXLRDIQRBLF(SEQ ID NO. 56) LSQEQLEHRERSXQTXRDIQRBLF (SEQ ID NO. 57)LSQEQLEHRERSLXTLXDIQRBLF (SEQ ID NO. 58) LSQEQLEHRERSLQXLRXIQRBLF(SEQ ID NO. 59) LSQEQLEHRERSLQTXRDXQRBLF (SEQ ID NO. 60)LSQEQLEHRERSLQTLXDIXRBLF (SEQ ID NO. 61) LSQEQLEHRERSLQTLRXIQXBLF(SEQ ID NO. 62) LSQEQLEHRERSLQTLRDXQRXLF (SEQ ID NO. 63)LSQEQLEHRERSLQTLRDIXRBXF (SEQ ID NO. 64) LSQEQLEHRERSLQTLRDIQXBLXi, i + 3 double staples: (SEQ ID NO. 65) XSQXQLEHRERSLQTLRDIQXBLX(SEQ ID NO. 66) XSQXQLEHRERSLQTLRDIXRBXF (SEQ ID NO. 67)XSQXQLEHRERSLQTLRDXQRBXF i, i + 4 double staples: (SEQ ID NO. 68)XSQEXLEHRERSLQTLRDIXRBLX (SEQ ID NO. 69) XSQEXLEHRERSLQTLRDXQRBXF(SEQ ID NO. 70) XSQEXLEHRERSLQTLXDIQRXLF i, i + 7 double staples:(SEQ ID NO. 71) XSQEQLEXRERSLQTLXDIQRBLX (SEQ ID NO. 72)XSQEQLEXRERSLQTXRDIQRBXF (SEQ ID NO. 73) XSQEQLEXRERSLQXLRDIQRZLF.

In certain of claim 2, wherein the amino acid sequence of positions 351to 374 is selected from the following:

SEQ ID NO: 1: BCL9 HD2 domain: LSQEQLEHRERSLQTLRDIQRMLFSEQ ID NO: 2: BCL9 HD2 domain M372B LSQEQLEHRERSLQTLRDIQRBLFSEQ ID NO: 3 SAH-BCL9_(A): LSQEQLEHRERSLQTLRXIQRXLFSEQ ID NO: 4: SAH-BCL9_(B): LSQEQLEHRERSLXTLRXIQRBLFSEQ ID NO: 5: SAH-BCL9_(C): LSQEQLEHREXSLQXLRDIQRBLFSEQ ID NO: 6: SAH-BCL9_(B)(H358D): LSQEQLEDRERSLXTLRXIQRBLFSEQ ID NO: 7: SAH-BCL9_(B)(R359E): LSQEQLEHEERSLXTLRXIQRBLF

In another aspect, the invention provides a composition comprising thepeptide as described above and a pharmaceutically acceptable carrier.The invention further provides a peptide of the invention in apharmaceutical carrier in a unit dosage form.

In another aspect, the invention provides a method of inhibitingcanonical Wnt/β-catenin signaling in a subject, comprising administeringa peptide of the invention.

In another aspect, the invention provides a method of inhibiting bindingof BCL9 to β-catenin in a subject, comprising administering a peptide ofthe invention.

In one embodiment, the inhibition of binding of BCL9 to β-catenin iscaused by the structurally constrained peptide of the invention.

In another aspect, the invention provides a method of treating a diseaseor disorder mediated by BCL9/β-catenin binding in a subject, comprisingadministering to the subject a peptide of the invention.

In one embodiment, the subject has been identified as being in need ofan inhibitor of the BCL9/β-catenin interaction or Wnt signaling.

In another embodiment, the disease is cancer, tumor cell proliferation,tumor cell de-differentiation and metastasis, tumor migration, tumorinduced angiogenesis, cancer stem cell chemoresistance, and aproliferation disease; or involves wound healing, angiogenesis ordiabetes.

The invention provides methods for the amelioration or treatment ofcancer, for example in a subject, by administration of astructurally-constrained peptide of the invention to the subject in atherapeutically effective amount. The method can further include one ormore of identifying a subject as being in need of amelioration ortreatment of cancer, or monitoring the subject for the prevention,amelioration, or treatment of cancer. In certain embodiments, theinvention provides methods of amelioration or treatment of cancer wherein the subject is identified as being in need of BCL9/β-cateninmodulation.

In a further embodiment, the disease is colorectal cancer, multiplemyeloma, lung cancer, colon cancer, breast cancer, prostate cancer,liver cancer, pancreas cancer, brain cancer, kidney cancer, ovariancancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breastcancer, pancreatic cancer, glioma, glioblastoma, hepatocellularcarcinoma, papillary renal carcinoma, head and neck squamous cellcarcinoma, leukemias, lymphomas, myelomas, and solid tumors.

In another aspect, the invention provides a method of treating cancer ina subject, comprising administering to the subject a peptide of theinvention.

In one embodiment, the subject has been previously identified as in needof a canonical Wnt/β-catenin signaling inhibitor to treat the cancer.

In another embodiment, the disease involves wound healing, angiogenesisor diabetes.

In other embodiments, the subject is administered with an additionaltherapeutic agent, radiation or chemotherapy.

In a further embodiment, the additional therapeutic compound is ananti-cancer compound.

In another further embodiment, the compound and the additionaltherapeutic agent are administered simultaneously or sequentially.

In other embodiments, the compound and the additional therapeutic agentare linked together (ie. a bifunctional compound).

In certain embodiments, the subject is a human.

In another aspect, the invention provides a kit comprising astructurally constrained peptide of the invention and instructions foruse in treating cancer.

In another aspect, the invention provides a method of identifying acompound that inhibits binding of BCL9 to β-catenin, comprising thesteps of contacting the peptide of the invention with β-catenin and thenscreening for small molecules or compounds that disrupt the interactionbetween the peptide of the invention and β-catenin.

Other embodiments of the invention will be understood base on thedisclosure provided infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1I show the design, synthesis, and characterization ofSAH-BCL9 peptides. FIG. 1A shows the alpha-helical HD2 domain of BCL9,which directly engages a surface groove of β-catenin, provided thetemplate for structural stabilization by hydrocarbon stapling. FIG. 1Bshows that SAH-BCL9 A-C(SEQ ID NOS 3-5, respectively) were generated byreplacing native residues on the non-interacting surface of the HD2domain with olefinic non-natural amino acids, which when subjected toolefin metathesis yield the corresponding stapled peptides. Anunmodified template peptide (BCL9 HD2 (SEQ ID NO: 1)) was alsogenerated. FIG. 1C shows that CD analysis revealed marked alpha-helicalstabilization of SAH-BCL9 peptides compared to the unmodified templatepeptide. FIG. 1D shows that FITC-SAH-BCL9_(B) and β-cateninco-precipitated from the lysates of FITC-SAH-BCL9-treated Colo320 cellsby both anti-FITC and anti-β-catenin pulldown assays. TCL, totalcellular lysate. FIG. 1E shows that FITC-SAH-BCL9_(B) and β-cateninpreferentially localize to the nucleus of Colo320 cells as monitored byconfocal microscopy. FIG. 1F shows that FITC-SAH-BCL9_(B) and β-cateninpreferentially localize to the nucleus of Colo320 cells as monitored bycellular fractionation analyses. FIG. 1G shows that H358D and R359Ereverse polarity mutants of FITC-SAH-BCL9_(B) exhibited similar highpercent α-helicity compared to the wild-type SAH by CD analysis. FIG. 1His a bot showing point mutagenesis impaired FITC-SAH-BCL9/β-cateninco-immunoprecipitation, with the R359E-derivative serving as the mosteffective negative control. FIG. 1I shows that FITC-SAH-BCL9_(B) and itsR359E control exhibit dose-equivalent cellular uptake by Colo320 cells.

FIG. 2A-FIG. 2C show that SAH-BCL9_(B) disrupts nativeβ-catenin-BCL9/B9L complexes and represses Wnt/β-catenin/BCL9-driventranscription. FIG. 2A shows that SAH-BCL9_(B) dose-responsivelydisrupted the association of β-catenin with BCL9 and B9L, whereascellular treatment with SAH-BCL9_(B)(R359E) had no effect. Dissociationof β-catenin from BCL9/B9L correlated with the co-immunoprecipitation ofβ-catenin with FITC-SAH-BCL9_(B). FIG. 2B shows that Colo320 cells weretransfected with TOP-FLASH, incubated with vehicle or SAH-BCL9 peptidesand assayed for luciferase activity, which was normalized to Renillaluciferase control. SAH-BCL9_(B), but not SAH-BCL9_(B)(R359E), inhibitedWnt-dependent reporter activity. Error bars are mean+/−s.d. for assaysperformed in triplicate. *, P<0.01. FIG. 2C shows that qPCR analysisrevealed repression of the Wnt target genes VEGF, c-Myc and Axing, butnot GAPDH, in response to SAH-BCL9_(B) treatment. Vehicle andSAH-BCL9_(B)(R359E) had no effect. Error bars are mean+/−s.d. for assaysperformed in quadruplicate. *, P<0.01.

FIG. 3A-FIG. 3I show that SAH-BCL9_(B) blocks cellular proliferation,angiogenesis, and migration. FIG. 3A shows that SAH-BCL9_(B) treatmentreduced the growth of Colo320 and colorectal primary tumors (CCPT), asmonitored by [³H]-thymidine uptake at 24 h. *, P<0.01. FIG. 3C showsthat SAH-BCL9_(B), but not vehicle or SAH-BCL9_(B)(R359E), likewiseimpaired the growth of MM1S cells in the presence or absence ofWnt3A-conditioned medium (Wnt3A-CM) and multiple myeloma primary tumors(MMPT). *, P<0.01. FIG. 3D shows that SAH-BCL9_(B), but not vehicle orSAH-BCL9_(B)(R359E), likewise impaired the growth of MM1S cells in thepresence or absence of Wnt3A-conditioned medium (Wnt3A-CM) and multiplemyeloma primary tumors (MMPT). *, P<0.01. FIG. 3E shows thatSAH-BCL9_(B) had no effect on the viability of Colo320 and MM1S cells asassessed by CellTiter-Glo at 72 h. FIG. 3F shows that correspondingly,SAH-BCL9_(B) did not activate caspase-3 or PARP, as monitored by westernanalysis. FIG. 3G shows that SAH-BCL9_(B) treatment decreased VEGFsecretion by Colo320 and MM1S cells as measured by ELISA. *, P<0.001FIG. 3H shows that HUVEC were cultured with supernatants collected fromColo320 or MM1S cells incubated with vehicle or SAH-BCL9_(B) peptidesand the number of tubes (black arrows) formed per high power fieldanalyzed by microscopy at 5 h. SAH-BCL9_(B) blocked in vitrocapillary-like tube formation. *, P<0.01 (n=3) FIG. 31 shows thatSAH-BCL9_(B) blocked the migration of Colo320 cells as monitored usingMatrigel Boyden chambers. Vehicle and SAH-BCL9_(B)(R359E) had no effect.*, P<0.01. Vehicle, 0.5% DMSO; SAH-BCL9 peptides, 5 μM. Error bars aremean+/−s.d. for experiments performed in triplicate.

FIG. 4A-FIG. 4I show that SAH-BCL9_(B) inhibits tumor growth,angiogenesis, and metastasis in vivo. FIG. 4A shows that cohorts (n=6)of NOD/SCID mice bearing intraperitoneal GFP-positive Colo320 cells weretreated with vehicle (2.5% DMSO in D5W) or SAH-BCL9_(B) peptides (20mg/kg) administered intraperitoneally every other day for a total of sixdoses. Whole body imaging and necropsy revealed decreased tumor burdenand liver metastases in SAH-BCL9_(B)-treated mice but not in vehicle-and SAH-BCL9_(B)(R359E)-treated animals. FIG. 4B shows that histologicanalysis of the liver revealed decreased tumor invasion andCD44-positivity in SAH-BCL9_(B)-treated mice. FIG. 4C shows the totalnumber of intraparenchymal nodules for each experimental group (n=6), asquantified by examining liver sections at 5 mm intervals, was markedlydecreased in SAH-BCL9_(B)-treated mice. Error bars are mean+/−s.d. *,P<0.01. FIG. 4D shows that SAH-BCL9_(B) treatment likewise inhibitedangiogenesis as evaluated by tumor blood vessel quantitation andanti-CD34 immunostaining. Error bars are mean+/−s.d. *, P<0.0001 (n=6).FIG. 4E shows that SAH-BCL9_(B) treatment likewise inhibitedangiogenesis as evaluated by tumor blood vessel quantitation andanti-CD34 immunostaining. Error bars are mean+/−s.d. *, P<0.0001 (n=6).FIG. 4F shows that cohorts of SCID-hu mice (n=5) bearing human bonechips populated by GFP-positive INA-6 cells were injected locally withvehicle (2.5% DMSO in D5W) or SAH-BCL9_(B) peptides (5 mg/kg) everyother day for a total of ten doses. Tumor burden was evaluated byshuIL-6R serum levels at the indicated days after injection of tumorcells. FIG. 4G shows that the cohorts of SCID-hu mice (n=5) bearinghuman bone chips populated by GFP-positive INA-6 cells were injectedlocally with vehicle (2.5% DMSO in D5W) or SAH-BCL9_(B) peptides (5mg/kg) every other day for a total of ten doses. Tumor burden wasevaluated by shuIL-6R serum levels at the indicated days after injectionof tumor cells. SAH-BCL9_(B) treatment significantly suppressed shuIL-6Rproduction and tumor burden, as reflected by decreased bone chipfluorescence. FIG. 4H shows that the histologic analysis likewisedemonstrated substantial reduction of INA-6 cells in the bone chips ofSAH-BCL9_(B)-treated mice, with tumor cells confined to the bone. Invehicle- and SAH-BCL9_(B)(R359E)-treated mice, tumor cells migratedoutside of the bone chip and invaded adjacent soft tissue (blackarrows). FIG. 4I shows that intratumoral angiogenesis was suppressed inSAH-BCL9_(B)-treated mice, as monitored by blood vessel quantitation andanti-CD34 immunostaining.

FIG. 5 shows targeting Wnt transcriptional activity in cancer usingStabilized Alpha-Helices of BCL9. Deregulated Wnt signaling underliesthe pathogenesis of a broad range of human cancers yet the developmentof targeted therapies to disrupt the pathway has remained a challenge.β-catenin is a central effector of the canonical Wnt pathway, activatingthe expression of genes such as c-Myc, cyclin D1, VEGF, and CD44 thatare involved in cell proliferation, migration, and angiogenesis. BCL9 isan important co-activator for β-catenin-mediated transcription, and ishighly expressed in tumors but not in the cells of origin, presenting anopportunity to selectively inhibit pathologic β-catenin activity. Guidedby the structure of the BCL9/β-catenin complex, Stabilized Alpha-Helicesof BCL9 (SAH-BCL9) were generated to block Wnt signaling in cancerthrough targeted disruption of the BCL9/β-catenin complex. SAH-BCL9reduces Wnt transcriptional activity and the expression of Wnt/β-catenintranscriptional targets, impeding tumor cell proliferation, migration,invasion, and angiogenesis in vitro and in vivo.

FIG. 6A-FIG. 6B show that BCL9 is overexpressed in a broad range oftumors. FIG. 6A shows that immunohistochemical studies performed ontissue microarrays from colon (n=23), breast (n=22), lung (n=32), liver(n=29), and ovarian (n=32) carcinomas revealed high levels of BCL9expression in a wide variety of tumors. Representative cases of tumorswith high or low level BCL9 immunostaining are shown. FIG. 6B shows thatblocking experiments using the immunizing BCL9 peptide (Abcam) wereperformed on human colorectal cancer specimens according to themanufacturer's protocol and documented BCL9 antibody specificity.

FIG. 7A-FIG. 7B show equivalent cellular uptake of SAH-BCL9 peptides.FIG. 7A shows that FITC-SAH-BCL9_(B) and FITC-SAH-BCL9_(B)(R359E)peptides exhibit equivalent temperature-dependent uptake in Colo320cells, consistent with the energy-dependent endocytic uptake mechanismpreviously documented for stapled peptides (Bernal, F., et al. J Am ChemSoc 129, 2456-7 (2007); Walensky, L. D. et al. Science 305, 1466-70(2004)). FIG. 7B shows that FITC-SAH-BCL9_(B) andFITC-SAH-BCL9_(B)(R359E) peptides exhibit equivalent cellular uptake byColo320 and MM1S cells.

FIG. 8A-FIG. 8B show that SAH-BCL9_(B) selectively engages β-catenin anddoes not disrupt its interaction with a non-BCL9 binding partner. FIG.8A shows that importantly, β-catenin targeting by FITC-SAH-BCL9_(B) isselective for disruption of the BCL9/β-catenin complex and does notaffect anti-β-catenin co-immunoprecipitation of E-cadherin from MCF7cell lysates. FIG. 8B shows that treatment of MCF7 cells withFITC-SAH-BCL9_(B) followed by anti-FITC pulldown performed on cellularlysates revealed the selective interaction between FITC-SAH-BCL9_(B) andβ-catenin, and no coimmunoprecipitation of unrelated proteins such asIxBα and actin. Single R359E point mutagenesis of the SAH-BCL9 bindinginterface abrogates co-immunoprecipitation of β-catenin, furtherconfirming the specificity of the SAH-BCL9_(B) peptide. MCF7 cells wereemployed in this assay as they contain readily detectable levels ofE-cadherin protein for monitoring the β-catenin/E-cadherin interaction.

FIG. 9 shows that suppression of β-catenin/BCL9-driven transcription bySAH-BCL9_(B) is dose-responsively reversed by increased expression ofBCL9. HCT116 cells transfected with TOP-FLASH and pcDNA-BCL9 weretreated with vehicle or SAH-BCL9 peptides (5 μM) and dual luciferaseassays were performed at 24 h. The suppression of reporter activities bySAH-BCL9_(B) is dose-responsively reversed by increasing BCL9 proteinexpression, highlighting the on-target specificity of SAH-BCL9_(B)-basedinhibition of β-catenin/BCL9-driven transcription. *P<0.01. HCT116 cellswere employed in this assay due to their relatively low level expressionof endogenous BCL9.

FIG. 10 shows inhibition of Wnt-specific transcriptional reporteractivity by SAH-BCL9_(B). SAH-BCL9_(B) dose-responsively blocked dGFPexpression under the transcriptional control of TCF regulatory sequences(7×TdG) in Colo320 cells. In contrast, treatment withSAH-BCL9_(B)(R359E) had little to no effect.

FIG. 11A-FIG. 11B show that VEGF is a direct transcriptional target ofBCL9. FIG. 11A shows that chromatin immunoprecipitation analysis (ChIP)of Colo320 cells using anti-TCF-4, β-catenin, and BCL9 antibodiesdocumented that, like c-Myc, the VEGF promoter is a target of theWnt/β-catenin/BCL9 transcriptional complex. Negative controls includedChIP with IgG and the use of primers to a non-specific, upstream regionof the VEGF promoter. FIG. 11B shows that Colo320 cells lentivirallytransduced with control shRNA or BCL9 shRNA vector were transfected withVEGF promoter-luciferase reporter plasmids. Reporter activity wasassayed using the dual luciferase assay system and results normalized toRenilla values for each sample. BCL9 knockdown effectively decreasedtranscriptional activity at the VEGF promoter. *, P<0.001.

FIG. 12 shows normal appearance of colonic mucosa and bone marrow inSAH-BCL9-treated mice. H&E staining of colonic mucosa and bone marrowtissues isolated from experimental mice treated with vehicle orSAH-BCL9-peptides showed no evidence of toxicity across all histologicspecimens.

FIG. 13A-FIG. 13B show that SAH-BCL9_(B) inhibits the proliferation ofINA-6 multiple myeloma cells. FIG. 13A shows that expression of BCL9 andβ-catenin proteins in MM1S and INA-6 cells, as detected by westernanalysis. FIB. 13B shows that INA-6 cells exposed to SAH-BCL9_(B) (5 μM)for 24 h displayed significantly reduced growth compared to vehicle- andSAH-BCL9_(B)(R359E)-treated cells, as measured by thymidineincorporation. *, P<0.001. Error bars are mean+/−s.d. for assaysperformed in triplicate.

FIG. 14 shows examples of non-natural olefinic amino acids inserted intopeptide templates to generate hydrocarbon-stapled peptides by olefinmetathesis.

FIG. 15A-FIG. 15D show examples of singly-, doubly-, and sequentiallystapled BCL9 HD2 peptides. X, stapling amino acid; B, norleucine. Astaple scan readily enables iterative production and testing of distinctstaple compositions and their differential positions along the peptidesequence to identify optimally stabilized alpha-helix of BCL9 HD2constructs. FIG. 15A-FIG. 15D discloses SEQ ID NO: 2, residues 2-6 ofSEQ ID NO:2 and SEQ ID NOS 8-115, respectively, in order of appearance.

FIG. 16 shows that three dimensional structure of residues 351-374 ofthe α-helical HD2 domain of BCL9 (SEQ ID NO. 140), highlighting thoseamino acid residues of the BCL9 HD2 interaction face that contactβ-catenin. FIG. 16 discloses residues 1-4 of SEQ ID NO: 140.

FIG. 17A-FIG. 17B show that SAH-BCL9_(B) disrupts β-catenin-BCL9/B9Lcomplexes. FIG. 17 A shows that differential binding affinities ofSAH-BCL9_(B) and SAH-BCL9_(B)(R359E) for recombinant β-catenin. FIG. 17Bshows that SAH-BCL9_(B) dose-responsively dissociated recombinantβ-catenin/BCL9 complexes as demonstrated by GST-pull-down assay. R359Epoint mutagenesis reduced SAH-BCL9_(B) activity by 6-fold.

FIG. 18A-FIG. 18F show that SAH-BCL9_(B) selectively blocks Wnttranscription. FIG. 18A shows that qRT-PCR analysis revealeddose-dependent repression of Wnt target genes in response toSAH-BCL9_(B) treatment of Colo320 cells. Error bars are mean+/−s.d. forassays performed in quadruplicate. * p<0.01. FIG. 18B shows quantitativecomparison of genes down-regulated by SAH-BCL9_(B) and dominantnegativeTCF1/TCF4 expression in DLDI cells across adenoma signatures. FIG. 18Cis a schematic showing quantitative comparison of genes down-regulatedby SAH-BCL9_(B) and dominant-negative TCF1/TCF4 expression in DLDI cellsacross carcmoma signatures. FIG. 18D shows heat map representation ofthe 50 most down regulated genes (p<0.001) of the leading edge—the genescontributing most to the correlation between SAH-BCL9_(B) anddominant-negative TCF1/TCF4, for the adenoma signatures. FIG. 18E is aschematic heat map representation of the 50 most down regulated genes(p<0.001) of the leading edge—the genes contributing most to thecorrelation between SAH-BCL9_(B) and dominant-negative TCF1/TCF4, forthe carcinoma signatures. FIG. 18F shows qRT-PCR validation of key Wnttarget genes in DLD1 cells treated with SAH-BCL9_(B).

FIG. 19A-FIG. 19B show the suppression of Wnt target gene expression bySAH-BCL9_(B). FIG. 19A shows that qRT-PCR analysis revealed repressionof Wnt target genes in response to SAH-BCL9_(B) treatment of MM1S cellsat 10 μM. Error bars are mean+/−s.d. for assays performed inquadruplicate. * p<0.01. FIG. 19B shows that Affymetrix gene expressionprofiling analysis of VEGF-A in DLD1 cells.

FIG. 20A-FIG. 20B show that SAH-BCL9_(B) selectively inhibitsproliferation of cultured colon cancer cells that are driven bypathologic Wnt signaling and express BCL9. FIG. 20A shows thatSAH-BCL9_(B), but not vehicle or SAH-BCL9_(MUT), significantly reducedthe proliferation of CRC cell lines. SAH-BCL9_(B)-susceptible cancercells express BCL9, whereas the LS174T cell line that does not expressBCL9 showed no response, linking the inhibitory effect of SAH-BCL9_(B)with BCL9 expression. As further cellular controls for SAH-BCL9_(B)'sspecificity-of-action, HCT116 cells and its two derivative cell lines,HCT116DO20 and HCT116KO58, whose proliferative capacity does not dependon Wnt/β-catenin activity (T. A. Chan et al., Proc Natl Acad Sci USA 99,8265 (2002)), showed no sensitivity to SAH-BCL9_(B). FIG. 20B shows theexpression of BCL9, B9L, and β-catenin in CRC cell lines, as evaluatedby western blot.

FIG. 21A-FIG. 21B show that SAH-BCL9_(B) enhances the cytotoxic effectof conventional chemotherapeutic agents. FIG. 21A shows that Colo320cells were co-cultured with vehicle, SAH-BCL9_(B), or SAH-BCL9_(MUT) andincreasing concentrations of 5-fluorouracil (5-FU), and evaluated forcellular proliferation using ³H-thymidine incorporation. FIG. 21B showsthat MM1S cells were co-cultured with vehicle, SAH-BCL9_(B), orSAH-BCL9_(MUT) and increasing concentrations of doxorubicin (Dox), andevaluated for cellular proliferation using ³H-thymidine incorporation.SAH-BCL9_(B) but not SAH-BCL9_(MUT) or vehicle significantly enhancedthe anti-tumor activity of the conventional agents.

FIG. 22 shows increased apoptosis in colonic tumor tissue ofSAH-BCL9_(B)-treated mice. Tumor tissue of NOD/SCID mice bearingintraperitoneal Colo320 cells were evaluated for apoptosis inductionusing TUNEL assay (brown). SAH-BCL9_(B), but not vehicle orSAH-BCL9_(MUT), notably increased TUNEL positivity. Three representative40× power fields are shown, including quantitation of TUNEL positivityin 6 high power fields. * p<0.001.

FIG. 23A FIG. 23B show that proliferation of INA-6 cells is dependent onWnt transcriptional activity and increased apoptosis in myeloma tumortissue of SAH-BCL9_(B)-treated mice. FIG. 23A shows that INA-6 cellswere lentivirally transduced with empty vector (Mock) or a vectorexpressing a dominant negative form of TCF4 (EdTP) and proliferation wasmeasured by ³H-thymidine incorporation. FIG. 23B shows tumor tissuesections from NOD/SCID mice bone chips bearing INA-6 cells wereevaluated for apoptosis induction using TUNEL assay (brown).SAH-BCL9_(B), but not vehicle or SAH-BCL9_(MUT), notably increased TUNELpositivity. Three representative 40× power fields are shown, includingquantitation of TUNEL positivity in 6 high power fields. * p<0.001.

DETAILED DESCRIPTION

Recent studies have revealed that high Wnt signaling activity isfunctionally ascribed to the colon cancer stem cell (CSC) population,which is resistant to conventional chemotherapy and believed to beresponsible for tumor recurrence (Vermeulen L, et al. Nat Cell Biol.12:468, 2010). Thus, blocking Wnt signaling pathway may be most potentagainst these cells. Furthermore canonical Wnt pathway has beenimplicated in physiological an pathological angiogenesis (Dejana E.Circulation Research. 107:943 2010), underscoring the relevance of thispathway for target drug discovery and therapeutic development (Barker,N., & Clevers, H. Nat Rev Drug Discov 5:997, 2006).

The crystal structure of the β-catenin/BCL9/TCF-4 complex revealed thatthe BCL9 binding site on β-catenin is distinct from other bindingpartners in that the α-helical HD2 domain of BCL9 (residues 352-374)binds a surface groove formed by α-helices 2 and 3 of the armadillorepeat 1 of β-catenin (FIG. 1A) (Sampietro, J. et al. Mol Cell 24,293-300 (2006)). Importantly, alanine mutagenesis of key residues at theBCL9 binding interface, such as H358A or R359A, blocked the ability ofBCL9 to bind β-catenin, abrogating transactivation. To harness thisnatural binding motif to target β-catenin, hydrocarbon stapling wasapplied (Schafmeister, C., Po, J. & Verdine, G. J Am Chem Soc 122,5891-5892 (2000); Walensky, L. D. et al. Science 305, 1466-70 (2004)) togenerate structurally-reinforced α-helical peptides based on the BCL9HD2 domain. Non-natural amino acids with olefinic side chains weresubstituted at (i, 1+4) positions followed by ruthenium-catalyzed olefinmetathesis to yield SAH-BCL9 peptides A-C (FIG. 1B). Circular dichroism(CD) analysis confirmed that hydrocarbon stapling consistently enhancedpeptide α-helicity compared to the corresponding unmodified peptide(BCL9 HD2) (FIG. 1C). Cells treated with fluorescent derivatives of thepeptides, followed by washing, trypsinization, and extraction, containedFITC-SAH-BCL9 A-C but not the corresponding unmodified FITC-peptide inthe lysates, documenting the cellular uptake of SAH peptides (FIG. 1D).Both FITC and β-catenin immunoprecipitation identified SAH-BCL9_(B) asthe most effective β-catenin interactor in situ (FIG. 1D).Immunofluorescence confocal microscopy demonstrated a predominantnuclear localization of β-catenin and SAH-BCL9_(B) in the nucleus (FIG.1E), with the nuclear enrichment of SAH-BCL9_(B) confirmed by cellularfractionation (FIG. 1F). To develop a negative control SAH-BCL9_(B)peptide for biological studies, SAH-BCL9_(B)(H358D) andSAH-BCL9_(B)(R359E) were generated and contain single reverse polaritypoint mutants of key binding interface residues (FIG. 1A). Compared toSAH-BCL9_(B), both mutants displayed similar α-helical enhancement (FIG.1G) and cellular uptake (FIG. 1H), yet demonstrated impaired β-catenininteraction by co-immunoprecipitation analysis (FIG. 1H), with R359Emutagenesis causing the most deleterious effect. Thus, SAH-BCL9_(B) andits corresponding R359E mutant were selected for functional studies inthe β-catenin/BCL9-dependent cell lines Colo320 and MM1S (Mani, M. etal. Cancer Res 69, 7577-86 (2009); Ilyas, M., et al. Proc Natl Acad SciUSA 94, 10330-4 (1997)), which display dose-equivalent uptake of the twopeptides (FIG. 1I).

A series of co-immunoprecipitation analyses was performed to determineif I-catenin targeting by SAH-BCL9_(B) disrupted the endogenousinteractions of β-catenin with BCL9 and its close homologue B9L(Brembeck, F. H. et al. Genes Dev 18, 2225-30 (2004)), which contains anidentical HD2 domain (Sampietro, J. et al. Mol Cell 24, 293-300 (2006)).Strikingly, SAH-BCL9_(B), but not its R359E derivative, causeddose-responsive disruption of the β-catenin-BCL9/B9L complexes inanti-BCL9 and B9L co-immunoprecipitation studies (FIG. 2A).Correspondingly, FITC-SAH-BCL9_(B), but not SAH-BCL9_(B)(R359E),dose-responsively co-immunoprecipitated with β-catenin (FIG. 2A),linking β-catenin targeting by SAH-BCL9_(B) with disengagement of theI-catenin-BCL9/B9L complexes. Given the documented toxicities associatedwith agents that target β-catenin and broadly disrupt its proteininteractions, it was confirmed that FITC-SAH-BCL9_(B) had no effect onβ-catenin's homeostatic interaction with E-cadherin, consistent with thedistinct, non-overlapping location of the BCL9/β-catenin binding site.The target-based selectivity of FITC-SAH-BCL9_(B) was further documentedby anti-FITC pulldown, which co-precipitates β-catenin but not otherunrelated cellular proteins such as IxBα and actin.

To examine the functional consequences of SAH-BCL9_(B)-mediated complexdisruption, the effects of SAH-BCL9_(B) and SAH-BCL9_(B)(R359E) in aWnt-specific TCF reporter gene transcriptional assay were evaluated(Mani, M. et al., Cancer Res 69, 7577-86 (2009); Sustmann, C., et al.Mol Cell Biol 28, 3526-37 (2008)). Whereas SAH-BCL9_(B) treatmentreduced reporter activity by nearly 50%, vehicle and SAH-BCL9_(B)(R359E)showed no effect (FIG. 2B). Importantly, the specificity of the effectof SAH-BCL9 was documented by showing that the inhibitory effect ofSAH-BCL9 was selectively abrogated by transfection with increasingamounts of pcDNA-BCL9, and that SAH-BCL9_(B) had no effect on an NFκBreporter gene transcriptional assay (FIG. 2B). In a second Wnt-specificreporter assay that monitors dGFP, which is under the transcriptionalcontrol of TCF regulatory sequences, SAH-BCL9_(B), but not vehicle orSAH-BCL9_(B)(R359E), dose-responsively blocked dGFP expression.Quantitative PCR (qPCR) analysis was employed to measure the effects ofvehicle, SAH-BCL9_(B), and SAH-BCL9_(B)(R359E) on the expression ofβ-catenin/BCL9 target genes, including VEGF. SAH-BCL9_(B), but notvehicle or SAH-BCL9_(B)(R359E), significantly reduced mRNA levels ofVEGF, c-Myc, and Axing, but not GAPDH, a non-Wnt pathway target gene(FIG. 2C).

To examine the phenotypic consequences of pharmacologic disruption ofthe β-catenin/BCL9 complex, cellular proliferation, angiogenesis, andmigration assays were conducted. A consistent pattern emerged wherebySAH-BCL9_(B), but not vehicle or SAH-BCL9_(B)(R359E), reduced theproliferation of Colo320, MM1S, and primary CRC and MM cells (FIG.3A-FIG. 3D). Of note, SAH-BCL9_(B) treatment did not induce cell death,as evaluated by viability assays and western analysis for caspase-3 andPARP activation (FIG. 3E-FIG. 3F). To determine the effect ofSAH-BCL9_(B) on tumor cell-induced angiogenesis, Colo320 and MM1S cellswere treated with vehicle and SAH-BCL9_(B) peptides and then VEGF levelswere quantitated in the media. Consistent with the qPCR analysis, onlySAH-BCL9_(B) reduced the level of secreted VEGF (FIG. 3G). In an invitro angiogenesis assay, human umbilical vein endothelial cells (HUVEC)were cultured with supernatants from treated Colo320 or MM1S cells andthen scored for the formation of capillary tube-like formations bymicroscopy. HUVEC cells exposed to the supernatant fromSAH-BCL9_(B)-treated cells showed reduced capillary tube formationcompared to the vehicle- and SAH-BCL9_(B)(R359E)-treated controls (FIG.3H). SAH-BCL9_(B) also decreased the adhesive and invasive potential ofColo320 cells, as reflected by a significant reduction in the capacityof SAH-BCL9_(B)-treated cells to pass thorough the extracellular matrix,as evaluated using Matrigel-coated invasion chambers (FIG. 3I). Takentogether, these data demonstrate that SAH-BCL9_(B) specifically disruptsa series of physiologic processes regulated by the BCL9/β-catenintranscriptional complex.

To explore the therapeutic potential of targeting the BCL9/β-catenininteraction, the capacity of SAH-BCL9_(B) to suppress tumor growth wasexamined in vivo. GFP-expressing Colo320 cells (1×10⁶) were injectedinto the peritoneum of sublethally irradiated NOD/SCID mice. Two daysafter cellular injection, mouse cohorts (n=6) were treated with vehicle(2.5% DMSO in D5W), SAH-BCL9_(B), or SAH-BCL9_(B)(R359E) peptides (20mg/kg) for a total of 6 doses administered intraperitoneally every otherday. On day 40 of the experiment, mice were sacrificed and evaluated fortumor burden and metastasis by whole body imaging and histologicexamination of harvested GFP-positive tissues. Overall tissuefluorescence was markedly reduced in mice treated with SAH-BCL9_(B)compared to vehicle and SAH-BCL9_(B)(R359E)-treated animals (FIG. 4A).These data were consistent with an overall reduction of metastatic tumornodules observed in the livers of SAH-BCL9_(B)-treated mice (FIG.4B-FIG. 4C). Interestingly, tumor tissue from SAH-BCL9_(B)-treated micealso showed decreased tumor cell CD44 immunoreactivity (FIG. 4B), areduction in the number of intratumoral blood vessels (FIG. 4D), andless intense capillary CD34 immunoreactivity (FIG. 4E), suggesting thatSAH-BCL9_(B)-mediated suppression of tumor growth and metastasis mayderive at least in part from reduction of cell migration andangiogenesis. Importantly, no histologic changes in normal murinetissues were observed across the treatment groups upon necropsy.

In a second in vivo model, the impact of SAH-BCL9_(B) treatment on thegrowth of INA-6 MM cells within a human bone graft implanted in theflank of SCID-hu mice was examined (Tassone, P. et al. Blood 106, 713-6(2005)). GFP-labeled INA-6 cells (5×10⁶), which express both BCL9 andβ-catenin and are suppressed by SAH-BCL9_(B) in vitro were injected intobone grafts four weeks after implantation. Two days later, cohorts ofmice (n=5) were treated by local injection with vehicle (2.5% DMSO inD5W), SAH-BCL9_(B), or SAH-BCL9_(B)(R359E) peptides (5 mg/kg) for atotal of 10 doses administered every other day. To monitor tumor burden,the serum level of soluble human interleukin-6 receptor (shuIL-6R) wasmeasured, which is first detectable 3-4 weeks after INA-6 tumorengraftment. Whereas mice treated with vehicle- and SAH-BCL9_(B)(R359E)showed a progressive increase in shuIL-6R levels reflective of tumorgrowth, SAH-BCL9_(B)-treated mice maintained low to undetectable levelsthroughout the evaluation period (FIG. 4F). Mice were sacrificed 33 daysafter INA-6 cell injection and evaluated for MM tumor burden byfluorescence imaging, histologic analysis, and anti-CD34 staining.Consistent with the measured levels of shuIL-6R, tumor burden within thebone chip was significantly reduced in SAH-BCL9_(B)-treated mice (FIG.4G-FIG. 4H). Interestingly, the tumor cells present inSAH-BCL9_(B)-treated mice resided within the confines of the bone chip,whereas vehicle- and SAH-BCL9_(B)(R359E)-treated mice demonstratedinvasion into the surrounding soft tissue (FIG. 4H, black arrows).Similar to the CRC model, local angiogenesis was suppressed inSAH-BCL9_(B)-treated SCID-hu mice, as monitored by anti-CD34 stainingand blood vessel quantitation (FIG. 4I). Thus, in two distinct mousemodels of Wnt-driven cancer, SAH-BCL9 effectively suppressed tumorgrowth, invasion, and angiogenesis in a sequence-specific manner.

The β-catenin transcriptional complex is a high priority pharmacologictarget due to its pathologic role in a broad range of cancers. Becauseβ-catenin participates in a variety of homeostatic functions and engagesthe majority of its interaction partners using the same binding surface(Barker, N. & Clevers, H. Nat Rev Drug Discov 5, 997-1014 (2006)),achieving anti-cancer activity and selectivity remains a pressingchallenge. For example, PKF115-584, a small molecule identified byhigh-throughput screening for inhibitors of the β-catenin/TCFinteraction, blocked Wnt-specific transcriptional activity and reducedthe growth of colon cancer cells (Lepourcelet, M. et al. Cancer Cell 5,91-102 (2004)), but induced severe bone marrow hypoplasia, anemia, andgeneralized wasting of treated mice (Sukhdeo, K. et al. Proc Natl AcadSci USA 104, 7516-21 (2007)). Targeting the β-catenin-BCL9 interface asan alternate strategy is appealing because BCL9 (1) drives pathologicI-catenin transcriptional activity, (2) engages β-catenin at a uniquebinding site (Sampietro, J. et al. Mol Cell 24, 293-300 (2006)), and (3)is predominantly found in tumor tissue rather than the cells of origin(Mani, M. et al. Cancer Res 69, 7577-86 (2009)). Importantly,eliminating the BCL9/β-catenin interaction through genetic deletion ofBcl9 in a mouse model had no overt phenotypic consequences (Deka, J. etal. Cancer Res 70, 6619-28). Thus, hydrocarbon stapling was applied tostructurally-stabilize BCL9's α-helical HD2 domain that directly engagesβ-catenin. In doing so, it was determined that SAH-BCL9_(B) targetsβ-catenin in situ and selectively disrupts β-catenin-BCL9/B9L complexes.Pharmacologic blockade of these interactions coincided with inhibitionof β-catenin-dependent transcriptional activity and target geneexpression, and the suppression of tumor cell growth, angiogenesis, andmetastasis without overt damage to normal tissues. Thus, theseproof-of-principle experiments document that selective targeting of theβ-catenin-BCL9 interface in cancer is a promising strategy forinterrogating and combating oncogenic Wnt signaling.

The invention is based, at least in part, on the results providedherein, as well as PCT/US2009/000438 (WO 2009/108261; filed Jan. 23,2009) demonstrating that stabilized alpha helical peptides haveexcellent structural, proteolytic, acid, and thermal stability. It hasalso been determined that stabilized alpha helical peptides are highlyeffective in interfering with Wnt/β-catenin signaling, indicating thatthe peptide can be used for the treatment of cancer. Further, thestabilized alpha-helical peptides have superior pharmacologic propertiesin vivo compared to their unmodified counterparts, reducing thefrequency and quantity of stabilized alpha-helical peptide that needs tobe administered as compared to a native peptide sequence, and ensuringthat exposure is sustained.

From this point on, the term “stabilizing crosslink” or derivationthereof, shall refer to its namesake or other covalent, or ionic,crosslink such as, but not limited to, a disulfide, amide, ester,1,2,3-triazole, or other bioconjugate or biocompatible crosslink. Fromthis point on, the term “hydrocarbon-staple” or “hydrocarbon-crosslink”or derivation thereof, shall refer to its namesake or other hydrocarboncovalent, or ionic, bioconjugate or biocompatible crosslinks.

In the peptides provided herein, the alpha helix HD2 domain isstabilized with at least one molecular tether, e.g., hydrocarbon staple,but may include two, three or more hydrocarbon staples. The inclusion ofmultiple hydrocarbon staples is particularly suited for alpha helicalpeptides that are 16 or more amino acids in length. The inclusion ofmore than one (e.g., 2, 3, 4, 5, depending on the length of the peptide)hydrocarbon staples provides for exceptional proteolytic, structural,acid and thermal stability of the modified polypeptides, yieldingbioactive peptides with strikingly enhanced pharmacologic properties invivo (ref Bird et al, PNAS, 2010).

In the compounds provided herein, the HD2 domain is structurallyconstrained by one or more modifications of the native sequence. Thealpha-helix of the HD2 domain can be stabilized using a molecular tethersuch as a hydrocarbon staple. Alternatively, or in addition, amino acidsubstitutions can be made in or adjacent to the region to includenatural or non-natural amino acids to promote the desired structure ofthe peptide, to promote or maintain the desired angle between the twohelices or to orient the helices relative to each other, or to improvethe pharmacologic properties. In an embodiment, at least one of thehelices of the HD2 domain includes a molecular tether such as ahydrocarbon staple to promote or maintain the helical nature of thedomain. In another embodiment, both of the helices of the HD2 domaininclude a molecular tether(s) such as hydrocarbon staple(s) to promoteor maintain the helical nature of the domain.

Hydrocarbon Stapling of Polypeptides

Preferably the alpha helix or HD2 domain is stabilized with at least onehydrocarbon staple. Hydrocarbon staples suitable for use with any of themodified polypeptides are described herein and in U.S. Publication Nos.2005/0250680, 2010/0234563, 2007/0197772, 2006/0008848, 2006/0014675;U.S. Pat. Nos. 7,723,469, 7,192,713, and 7,084,244; InternationalPublication Nos. WO 2009/108261) and WO 2010/148335; and Kawamoto, S. A.et al., J. Med. Chem. 55, 1137-1146 (2012); Mahon, A. B. and Arora, P.S., Chem. Commun. 48, 1416-1418 (2012); and Chapman, R. N. et al., J.Am. Chem. Soc. 126, 12252-3 (2004), which are incorporated by referencein their entirety. Hydrocarbon stapling allows a polypeptide,predisposed to have a helical secondary structure, to maintain itsnative helical conformation and increase its stability and efficacy. Inone embodiment, the modified polypeptide has at least 10%, 20%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% or more helicity in an aqueoussolution as determined by circular dichroism. Assays for determiningcircular dichroism are known in the art and described herein.

The hydrocarbon stapled polypeptides include a tether (linkage) betweentwo amino acids, in which the tether significantly enhances the helicalsecondary structure of the polypeptide. Generally, the tether extendsacross the length of one or two helical turns (i.e., 3, 4 or 7 aminoacids). Accordingly, amino acids positioned at i and i+3; i and i+4; ori and i+7 are ideal candidates for chemical modification andcross-linking. Thus, any of the amino acid residues of the modifiedpolypeptides of the invention may be tethered (e.g., cross-linked) inconformity with the above. Suitable tethers are described herein and inU.S. Publication Nos. 2005/0250680, 2010/0234563, 2007/0197772,2006/0008848, 2006/0014675; U.S. Pat. Nos. 7,723,469, 7,192,713, and7,084,244; International Publication Nos. WO 2009/108261) and WO2010/148335; and Kawamoto, S. A. et al., J. Med. Chem. 55, 1137-1146(2012); Mahon, A. B. and Arora, P. S., Chem. Commun. 48, 1416-1418(2012); and Chapman, R. N. et al., J. Am. Chem. Soc. 126, 12252-3(2004). It is understood that tethers such as hydrocarbon staples can bepositioned at other intervals to promote helical variants (e.g., withdifferent pitches, angles, or residues and fractions thereof per turn)or structures other than helices.

In a further embodiment, the hydrocarbon staple(s) is positioned so asto link a first amino acid (i) and a second amino acid (i+3) which is 3amino acids downstream of the first amino acid. In another embodiment,the hydrocarbon staple links a first amino acid (i) and a second aminoacid (i+4) which is 4 amino acids downstream of the first amino acid. Inyet another embodiment, the hydrocarbon staple links a first amino acid(i) and a second amino acid (i+7) which is 7 amino acids downstream ofthe first amino acid.

The modified polypeptides of the invention will generally include thestructure of Formula (I), (II) or (III) provided below.

Any of the modified polypeptides described herein can be present in acomposition (e.g., pharmaceutical composition) or kit. In someembodiments of the invention, the composition or kit comprises two ormore modified polypeptides.

SAH-BCL9 Peptides

The modified polypeptides of the invention include the HD-2 peptidesamino acids 352 to 374 of the following

SEQ ID NO: 1: BCL9 HD2 domain: LSQEQLEHRERSLQTLRDIQRMLFSEQ ID NO: 2: BCL9 HD2 domain M372B: LSQEQLEHRERSLQTLRDIQRBLFSEQ ID NO: 3: SAH-BCL9_(A): LSQEQLEHRERSLQTLRXIQRXLFSEQ ID NO: 4: SAH-BCL9_(B): LSQEQLEHRERSLXTLRXIQRBLFSEQ ID NO: 5: SAH-BCL9_(C): LSQEQLEHREXSLQXLRDIQRBLFSEQ ID NO: 6: SAH-BCL9_(B)(H358D): LSQEQLEDRERSLXTLRXIQRBLFSEQ ID NO: 7: SAH-BCL9_(B)(R359E): LSQEQLEHEERSLXTLRXIQRBLF

Peptides corresponding to analogs of the full-length and truncated HD2domain or BCL9 peptides, described, above, may be contemplated by theinvention. The term “HD2 domain analogs”, as used herein, refers to apeptide that is recognized or identified as having a repeat-analogdomain or BCL9 domain. Methods for repeat-analog polypeptides are knownin the art, for example, bioinformatics programs based on pairwiseresidue correlations (e.g., on the world wide web at:ch.embnet.org/software/COILS_form.html), which have the ability torecognize coils from protein sequences and model their structures (SeeLupas, A., et al. Science 1991. 252: 1162-1164, which is incorporatedherein by reference). Further, such modified peptides exhibitanti-cancer activity. Methods for identifying BCL9 HD2 and other BCL9homologues are known in the art and can be performed using the criteriaset forth herein.

Mutations, Truncations, and Extensions of HD2 Domain and BCL9 Peptides

The amino acid substitutions may be of a conserved or non-conservednature. Conserved amino acid substitutions consist of replacing one ormore amino acids of the HD2 peptide sequence with amino acids of similarcharge, size, and/or hydrophobicity characteristics. Non-conservedsubstitutions consist of replacing one or more amino acids of the HD2domain sequence with amino acids possessing dissimilar charge, size,and/or hydrophobicity characteristics. Substitutions can include the useof conserved or non-conserved non-natural amino acids.

Amino acid insertions may consist of single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of the full-length or truncated HD2 domain or BCL9peptide, as well as at a position internal to the peptide. Suchinsertions will generally range from 2 to 15 amino acids in length. Itis contemplated that insertions made at either the carboxy or aminoterminus of the peptide of interest may be of a broader size range, withabout 2 to about 50 amino acids being preferred. One or more suchinsertions may be introduced into full-length or truncated HD2 domain orBCL9 peptide, as long as such insertions result in modified peptideswhich may still exhibit anti-cancer activity.

Deletions of full-length or truncated HD2 domain or BCL9 peptide arealso within the scope of the invention. Such deletions consist of theremoval of one or more amino acids from the HD2 domain or BCL9 peptide;or HD2 domain or BCL9 peptide-like peptide sequence, with the lowerlimit length of the resulting peptide sequence being 4, 5, or 6 aminoacids. Such deletions may involve a single contiguous or greater thanone discrete portion of the peptide sequences. One or more suchdeletions may be introduced into full-length or truncated HD2 domain orBCL9 peptide, as long as such deletions result in peptides which maystill exhibit anti-cancer activity.

Additionally, one skilled in the art would recognize that theinteraction between the hydrocarbon-stapled peptide and its targetprotein form a complex in which, on the peptide there can be defined, aninteracting face and a non-interacting face. Mutations along thenon-interacting face can be made facilely, whilst mutations on theinteracting face are not tolerated, such that the residues on theinteracting face are the main component of the complex and as suchshould be conserved and maintained in designed hydrocarbon-stapledpeptides. As such, greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90% of the residues on the interacting face of BCL9 HD2 domain in theβ-catenin/BCL9 complex should unchanged or changed to a structurally orchemically similar amino acid residue.

Stabilization of HD2 Domain and BCL9 Peptides

The modified polypeptides of the present invention are structurallyconstrained (e.g., stabilized, stapled) helical and/or include one ormore amino acid sequence modifications as compared to the native (i.e.,wild type or otherwise naturally occurring) sequence to incorporatenatural and/or non-natural amino acids to limit the structuralflexibility of the peptide as compared to the native sequence, whichloses bioactive shape when taken out of physiologic context. Preferably,the polypeptides include at least one molecular tether such as ahydrocarbon staple. Hydrocarbon stapling is described in U.S.Publication Nos. 2005/0250680, 2010/0234563, 2007/0197772, 2006/0008848,2006/0014675; U.S. Pat. Nos. 7,723,469, 7,192,713, and 7,084,244;International Publication Nos. WO 2009/108261) and WO 2010/148335; andKawamoto, S. A. et al., J. Med. Chem. 55, 1137-1146 (2012); Mahon, A. B.and Arora, P. S., Chem. Commun. 48, 1416-1418 (2012); and Chapman, R. N.et al., J. Am. Chem. Soc. 126, 12252-3 (2004), which are incorporatedherein by reference in their entirety.

The peptide α-helix participates in critically important proteininteractions by presenting specific amino acid residues in an orderedand precise arrangement over a relatively large contact surface area(Chittenden, T., et al., Embo Journal, 1995. 14(22): p. 5589-5596;Kussie, P. H., et al. Science, 1996. 274(5289): p. 948-953; Ellenberger,T. E., et al., Cell, 1992. 71(7): p. 1223-1237). Alpha-helical domainsand other protein structural features are frequently stabilized byscaffold sequences in the remainder of the protein, which facilitate theformation and/or maintenance of a helical structure, e.g., an α-helicalstructure. When taken out of context, α-helical peptide motifs canunfold, leading to loss of biological activity. Critical challenges indeveloping α-helical peptides include promoting and/or maintaining theirnatural α-helical structure and preparing peptides that can resistproteolytic, acid and thermal degradation, and thereby remain intact invivo.

Hydrocarbon stapling refers to a process for stably cross-linking apolypeptide via at least two substituted amino acids (or a non-nativelinkage, e.g., carbon-carbon, from two natural amino acids) that helpsto conformationally bestow the native secondary structure of thatpolypeptide. Hydrocarbon stapling promotes and maintains analpha-helical secondary structure in peptides that thermodynamicallyfavor an alpha-helical structure. This secondary structure increasesresistance of the polypeptide to proteolytic cleavage and heat, and alsomay increase hydrophobicity. Accordingly, the hydrocarbon stapled(structurally constrained, e.g., crosslinked) polypeptides describedherein have improved biological activity relative to a correspondingnon-hydrocarbon stapled (not structurally constrained) polypeptide. Thecross-linked polypeptides described herein can be used therapeutically,e.g., to treat cancer.

The hydrocarbon stapled polypeptides include a tether (linkage) betweentwo amino acids, which tether significantly enhances the helicalsecondary structure of the polypeptide. Generally, the tether extendsacross the length of one or two helical turns (i.e., about 3-3.6 orabout 7 amino acids). Accordingly, amino acids positioned at i and i+3;i and i+4; or i and i+7 are ideal candidates for chemical modificationand cross-linking. Thus, for example, where a peptide has the sequence .. . X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . , cross-links between X1and X4, or between X1 and X5, or between X1 and X8 are useful as arecross-links between X2 and X5, or between X2 and X6, or between X2 andX9, etc. The use of multiple cross-links (e.g., 2, 3, 4 or more) hasalso been achieved, compounding the benefits of individual stapledadducts (e.g., improved helicity and activity; improved helicity andthermal stability; improved helicity and acid stability; improvedhelicity and pharmacologic properties). The use of “stitched”cross-links has also been achieved whereby double linkages are made froma common origin (e.g., X1, X5, and X9, where X5 is the anchor point forboth staples). Thus, the invention encompasses the incorporation of oneor more crosslinks within the polypeptide sequence to either furtherstabilize the sequence or facilitate the structural stabilization,proteolytic resistance, thermal stability, acid stability, pharmacologicproperties, and biological activity enhancement of longer polypeptidestretches.

In some embodiments of the invention, the tethers, e.g., hydrocarbonstaples are used to stabilize structures other than helices. In suchcases, the ends of the tethers can be placed at intervals other than ati, i+3, i+4, and i+7.

In one embodiment, the modified polypeptides of the invention have theformula (I),

wherein;each R₁ and R₂ are independently H or a C₁ to C₁₀ alkyl, alkenyl,alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, orheterocyclylalkyl;R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n); each of which issubstituted with 0-6 R₅;R₄ is alkyl, alkenyl, or alkynyl;R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;n is an integer from 1-4;x is an integer from 2-10;each y is independently an integer from 0-100;z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); andeach Xaa is independently an amino acid.

The modified polypeptides may include an amino acid sequence which formsan alpha-helix and is 30% or more identical to, an amino acid sequenceof SEQ ID NO: 1-7; wherein X is any amino acid and further identifiesthe amino acid residues which are linked by a hydrocarbon staple, and Bis norleucine.

The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., C₅,C₈ or C₁₁ alkyl or a C₅, C₈ or C₁₁ alkenyl, or C₅, C₈ or C₁₁ alkynyl).The tethered amino acid can be alpha disubstituted (e.g., C₁-C₃ ormethyl).

In some instances, x is 2, 3, or 6.

In some instances, each y is independently an integer between 3 and 15.

In some instances each y is independently an integer between 1 and 15.

In some instances, R₁ and R₂ are each independently H or C₁-C₆ alkyl.

In some instances, R₁ and R₂ are each independently C₁-C₃ alkyl.

In some instances, at least one of R₁ and R₂ are methyl. For example R₁and R₂ are both methyl.

In some instances R₃ is alkyl (e.g., C₈ alkyl) and x is 3.

In some instances, R₃ is C₁₁ alkyl and x is 6.

In some instances, R₃ is alkenyl (e.g., C₈ alkenyl) and x is 3.

In some instances x is 6 and R₃ is C₁₁ alkenyl.

In some instances, R₃ is a straight chain alkyl, alkenyl, or alkynyl.

In some instances R₃ is —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—.

In certain embodiments the two alpha, alpha disubstituted stereocentersare both in the R configuration or S configuration (e.g., i, i+4cross-link), or one stereocenter is R and the other is S (e.g., i, i+7cross-link). Thus, where formula I is depicted as

the C′ and C″ disubstituted stereocenters can both be in the Rconfiguration or they can both be in the S configuration, for examplewhen X is 3. When x is 6, the C′ disubstituted stereocenter is in the Rconfiguration and the C″ disubstituted stereocenter is in the Sconfiguration. The R₃ double bond may be in the E or Z stereochemicalconfiguration.

In some instances R₃ is [R₄—K—R₄]_(n); and R₄ is a straight chain alkyl,alkenyl, or alkynyl.

In some embodiments the modified polypeptide comprises at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50,or more amino acids of a repeat or repeat like domain, e.g., a BCL9 HD2domain. Each [Xaa]_(y) is a peptide that can independently comprise atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 ormore amino acids of a repeat or repeat like domain, e.g., a HD2 domain.[Xaa]_(x) is a peptide that can comprise 3 or 6 amino acids of a repeator repeat like domain.

The modified polypeptide can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more aminoacids of a repeat or repeat like domain, e.g., a HD2 domain, e.g., apolypeptide having the amino acid sequence of SEQ ID NO: 1-7, whereintwo amino acids that are separated by two, three, or six amino acids arereplaced by amino acid substitutes that are linked via R₃. Thus, atleast two amino acids can be replaced by tethered amino acids ortethered amino acid substitutes. Thus, where formula (I) is depicted as

[Xaa]_(y′) and [Xaa]_(y″) can each comprise polypeptide sequences fromthe same or different heptad repeat or heptad repeat like domains.

The invention features cross-linked polypeptides comprising 10 (11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50or more) amino acids of a repeat or repeat like domain, e.g., a HD2domain e.g., a polypeptide having the amino acid sequence of SEQ ID NO:1-7 wherein the alpha carbons of two amino acids that are separated bytwo, three, or six amino acids are linked via R₃, one of the two alphacarbons is substituted by R₁ and the other is substituted by R₂ and eachis linked via peptide bonds to additional amino acids.

In another embodiment, the modified polypeptides of the invention havethe formula (II),

whereineach R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl; heteroarylalkyl; or heterocyclylalkyl;each n is independently an integer from 1-15;x is 2, 3, or 6each y is independently an integer from 0-100;z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);each Xaa is independently an amino acid.

In still another embodiment, the modified polypeptides of the inventionhave the formula (III),

wherein;each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or a naturally occurringamino acid side chain; each of which is substituted with 0-6 R₅;R₄ is alkyl, alkenyl, or alkynyl;R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

R₆ is H, alkyl, or a therapeutic agent;R₇ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or an naturally occurringamino acid side chain; each of which is substituted with 0-6 R₅;n is an integer from 1-4;x is an integer from 2-10;each y is independently an integer from 0-100;z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); andeach Xaa is independently an amino acid.

Also contemplated by the invention are the stitched peptides which aredisclosed at least at PCT/US2008/058575 (WO 2008/121767), the contentsof which are incorporated herein by reference.

The modified polypeptides may include an amino acid sequence that formsan alpha-helix and is 20% or more identical to, or contain at least 7amino acids from an amino acid sequence, or at least two amino acidsfrom a face of a helix formed by a peptide having the sequence of SEQ IDNO: 1-7; wherein X is any amino acid and further identifies the aminoacid residues which are linked by a hydrocarbon staple, and B isnorleucine. In certain embodiments, modified polypeptides may include anamino acid sequence that forms an alpha-helix and is 30% or moreidentical to a peptide having the sequence of SEQ ID NO: 1-7. In certainembodiments, the amino acid sequence in the alpha-helix is 40%, 50%,60%, 70%, 80%, 90%, or 95% or greater identical to a peptide having thesequence of SEQ ID NO: 1-7.

While hydrocarbon tethers have been described, other tethers are alsoenvisioned. For example, the tether can include one or more of an ether,thioether, ester, amine, or amide moiety. In some cases, a naturallyoccurring amino acid side chain can be incorporated into the tether. Forexample, a tether can be coupled with a functional group such as thehydroxyl in serine, the thiol in cysteine, the primary amine in lysine,the acid in aspartate or glutamate, or the amide in asparagine orglutamine. Accordingly, it is possible to create a tether usingnaturally occurring amino acids rather than using a tether that is madeby coupling two non-naturally occurring amino acids. It is also possibleto use a single non-naturally occurring amino acid together with anaturally occurring amino acid.

It is further envisioned that the length of the tether can be varied.For instance, a shorter length of tether can be used where it isdesirable to provide a relatively high degree of constraint on thesecondary structure, whereas, in some instances, it is desirable toprovide less constraint on the secondary structure, and thus a longertether may be desired. It is further understood that the insertion ofthe tether at a site or in an amino acid sequence when the amino acidsequence has no tendency to form a helix will not result in helixformation.

Additionally, while examples of tethers spanning from amino acids i toi+3, i to i+4; and i to i+7 have been described in order to provide atether that is primarily on a single face of the alpha helix, thetethers can be synthesized to span any combinations of numbers of aminoacids to promote and/or maintain the structures other than alphahelices.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the described herein will be evident to those ofordinary skill in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof. The specific method ofsynthesis of the peptides is not a limitation of the invention.

Synthesis of Peptides

The peptides of this invention can be made by chemical synthesismethods, which are well known to the skilled artisan and describedherein. See, for example, Fields et al., Chapter 3 in SyntheticPeptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York,N.Y., 1992, p. 77; and Bird, G. H., et al., Methods Enzymol 446, 369-86(2008). Hence, peptides can be synthesized using the automatedMerrifield techniques of solid phase synthesis with the alpha-NH₂protected by either t-Boc or Fmoc chemistry using side chain protectedamino acids on, for example, an Applied Biosystems Peptide SynthesizerModel 430A or 431 or the AAPPTEC multichannel synthesizer APEX 396.

One manner of making of the peptides described herein is using solidphase peptide synthesis (SPPS). The C-terminal amino acid is attached toa cross-linked polystyrene resin via an acid labile bond with a linkermolecule. This resin is insoluble in the solvents used for synthesis,making it relatively simple and fast to wash away excess reagents andby-products. The N-terminus is protected with the Fmoc group, which isstable in acid, but removable by base. Any side chain functional groupsare protected with base stable, acid labile groups.

Longer peptides can also be made by conjoining individual syntheticpeptides using native chemical ligation. Alternatively, longer syntheticpeptides can be synthesized by well known recombinant DNA techniques.Such techniques are provided in well-known standard manuals withdetailed protocols. To construct a coding sequence encoding a peptide ofthis invention, the amino acid sequence is reverse translated to obtaina nucleic acid sequence encoding the amino acid sequence, preferablywith codons that are optimum for the organism in which the gene is to beexpressed. Next, a coding sequence is made, typically by synthesizingoligonucleotides which encode the peptide and any regulatory elements,if necessary. The coding sequence is inserted in a suitable cloningvector and transfected into a host cell. Furthermore, the host cell isengineered so as to be able to incorporate the non-natural amino acidsfor the hydrocarbon staple. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. SeeLiu et al. Proc. Nat. Acad. Sci (USA), 94:10092-10097 (1997). Thepeptide is purified and characterized by standard methods.

The peptides can be made in a high-throughput, combinatorial fashion,e.g., using a high-throughput multichannel combinatorial synthesizersuch as that available from Advanced Chemtech/APPTTEC, Thuramed or CEM.

Definitions

An “agent” is understood herein to include a therapeutically activecompound or a potentially therapeutic active compound. An agent can be apreviously known or unknown compound. As used herein, an agent istypically a non-cell based compound, however, an agent can include abiological therapeutic agent, e.g., peptide or nucleic acid therapeutic,cytokine, etc.

As used herein “amelioration” or “treatment” is understood as meaning tolessen or decrease at least one sign, symptom, indication, or effect ofa specific disease or condition. Amelioration and treatment can requirethe administration of more than one dose of an agent, either alone or inconjunction with other therapeutic agents and interventions.Amelioration or treatment do not require that the disease or conditionbe cured.

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the 20 common naturallyoccurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G,H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letterabbreviations)) as well as the naturally occurring and non-naturallyoccurring amino acids including beta-amino acids and α,α disubstitutedamino acids, prepared by organic synthesis or other metabolic routes andthat can be applied for specialized uses such as increasing chemicaldiversity, functionality, binding capacity, structural mimesis, andstability.

The term “amino acid side chain” or “amino acid R group” refers to amoiety attached to the α-carbon in an amino acid. For example, the aminoacid side chain or R group for alanine is methyl, the amino acid sidechain for phenylalanine is phenylmethyl, the amino acid side chain forcysteine is thiomethyl, the amino acid side chain for aspartate iscarboxymethyl, the amino acid side chain for tyrosine is4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acidside chains are also included, for example, those that occur in nature(e.g., an amino acid metabolite) or those that are made synthetically(e.g., an alpha, alpha di-substituted amino acid, a beta-amino acid).

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of the analyte or diagnostic or therapeuticindicator to be detected at a level that is statistically different thana sample from a normal, untreated, or control sample. Control samplesinclude, for example, cells in culture, one or more laboratory testanimals, or one or more human subjects. Methods to select and testcontrol samples are within the ability of those in the art. An analytecan be a naturally occurring substance that is characteristicallyexpressed or produced by the cell or organism or a substance produced bya reporter construct (e.g., β-galactosidase or luciferase). Depending onthe method used for detection the amount and measurement of the changecan vary. Determination of statistical significance is within theability of those skilled in the art.

“Co-administration” as used herein is understood as administration ofone or more agents to a subject such that the agents are present andactive in the subject at the same time. Co-administration does notrequire a preparation of an admixture of the agents or simultaneousadministration of the agents.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. For example, families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Other conserved amino acid substitutions can also occuracross amino acid side chain families, such as when substituting anasparagine for aspartic acid in order to modify the charge of a peptide.Thus, a predicted amino acid residue in a HD2 domain peptide, forexample, is preferably replaced with another amino acid residue from thesame side chain family or homologues across families (e.g., asparaginefor aspartic acid, glutamine for glutamic acid). Conservative changescan further include substitution of chemically homologous non-naturalamino acids (i.e., a synthetic non-natural hydrophobic amino acid inplace of leucine, a synthetic non-natural aromatic amino acid in placeof tryptophan).

“Contacting a cell” is understood herein as providing an agent to a testcell e.g., a cell to be treated in culture or in an animal, such thatthe agent or isolated cell can interact with the test cell or cell to betreated, potentially be taken up by the test cell or cell to be treated,and have an effect on the test cell or cell to be treated. The agent orisolated cell can be delivered to the cell directly (e.g., by additionof the agent to culture medium or by injection into the cell or tissueof interest), or by delivery to the organism by an enteral or parenteralroute of administration for delivery to the cell by circulation,lymphatic, or other means.

As used herein, “detecting”, “detection” and the like are understoodthat an assay performed for identification of a specific analyte in asample, a product from a reporter construct in a sample, or an activityof an agent in a sample.

By “diagnosing” as used herein refers to a clinical or other assessmentof the condition of a subject based on observation, testing, orcircumstances for identifying a subject having a disease, disorder, orcondition based on the presence of at least one sign or symptom of thedisease, disorder, or condition. Typically, diagnosing using the methodof the invention includes the observation of the subject for other signsor symptoms of the disease, disorder, or condition.

The terms “effective amount,” or “effective dose” refers to that amountof an agent to produce the intended pharmacological, therapeutic orpreventive result. The pharmacologically effective amount results in theamelioration of one or more signs or symptoms of a disorder providedherein, or prevents the spread of the disorder. For example, atherapeutically effective amount preferably refers to the amount of atherapeutic agent that decreases the rate of cancer spread, by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or more as compared to an untreatedcontrol subject. More than one dose of an agent may be required toprovide an effective dose.

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. On theother hand, the term “ineffective” indicates that a treatment does notprovide sufficient pharmacological effect to be therapeutically useful,even in the absence of deleterious effects, at least in the unstratifiedpopulation. (Such a treatment may be ineffective in a subgroup that canbe identified by the expression profile or profiles.) “Less effective”means that the treatment results in a therapeutically significant lowerlevel of pharmacological effectiveness and/or a therapeutically greaterlevel of adverse physiological effects.

Thus, in connection with the administration of a drug, a drug which is“effective against” a disease or condition indicates that administrationin a clinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such as aimprovement of symptoms, a cure, a reduction in disease signs orsymptoms, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating the particular type of disease or condition.

As use herein, the “face” of a helix, for example, an alpha-helix or a3₁₀ helix, is understood as the amino acids that are “stacked” in ahelix of a protein so that when the helix is positioned vertically, theamino acids in a single face are depicted as being one on top of theother. For example, an alpha-helix has about 3.6 amino acids per turn.Therefore, when a peptide having a sequence abcdefga′b′c′d′e′f′g′ formsan alpha helix, the fourth and fifth amino acids (i+3 and i+4), i.e.,amino acids d and e, will “stack” over the first amino acid (position1+˜3. 6 amino acids), and the eighth amino acid, amino acid a′ (i+7),will stack over amino acid a to form a face of the helix and starting anew turn with amino acid a′. In an alpha-helix, amino acid b, the secondamino acid, will “stack” with the fifth and sixth amino acids, i.e.,amino acids e and fat the +3 and +4 positions, and with amino acid b′ atthe +7 position to form a face of the helix. Faces on helices startingwith amino acid c, d, e, f, and g can be readily determined based on theabove disclosure. Furthermore, a face of a helix can include twoadjacent, three adjacent, or four adjacent columns of “stacked”residues.

An example of a “face” of a helix includes the “interacting face” of thehelix. An “interacting face” amino acid residue is a residue that makescontact with one or more helices in the helix bundle, results inabolishing or substantially abolishing the polypeptide functionalactivity. Substantially abolishing is understood as reducing thefunctional activity of a BCL9 peptide to less than about 50%, less thanabout 40%, less than about 30% of the wild-type peptide in anappropriate assay. The interacting face amino acid residues of the BCL9peptides can readily be determined by methods well known in the art andare described herein. In one embodiment, an essential amino acid residueis in the “a” or “d” position of a BCL9 HD2 domain, while non-essentialamino acids may occur in a “b”, “c”, “e”, “f” or “g” position. The term“interacting face” amino acid residue as used herein, includesconservative substitutions of the interacting face amino acids that donot disrupt function of the sequence. Generally, the “interacting face”amino acid residues are found at the interacting face of the alphahelix.

The BLC9, BCL9-like, and HD2 domain and HD2 domain analogs are readilyidentifiable by those possessing ordinary skill in the art by sequencebased homology, structural homology and/or functional homology. Suchmethods are well known in the art and include bioinformatics programsbased on pairwise residue correlations (e.g.,ch.embnet.org/software/COILS_form.html), which have the ability torecognize coils from protein sequences and model their structures (SeeLupas, A., et al. Science 1991. 252(5009); p. 1162-1164).

In one embodiment, the modified polypeptide of the invention is 20% ormore similar at the interacting face to the amino acid sequence of SEQID NO:1-7. In another embodiment, the modified polypeptide of theinvention is 30% or more similar at the interacting face to the aminoacid sequence of SEQ ID NO:1-7. In another embodiment, the modifiedpolypeptide of the invention is 40% or more similar at the interactingface to the amino acid sequence of SEQ ID NO:1-7. In another embodiment,the modified polypeptide of the invention is 50% or more similar at theinteracting face to the amino acid sequence of SEQ ID NO:1-7. In anotherembodiment, the modified polypeptide of the invention is 60% or moresimilar at the interacting face to the amino acid sequence of SEQ IDNO:1-7. In another embodiment, the modified polypeptide of the inventionis 70% or more similar at the interacting face to the amino acidsequence of SEQ ID NO:1-7. In another embodiment, the modifiedpolypeptide of the invention is 80% or more similar at the interactingface to the amino acid sequence of SEQ ID NO:1-7. In another embodiment,the modified polypeptide of the invention is 90% or more similar at theinteracting face to the amino acid sequence of SEQ ID NO:1-7. Theinteracting face of BCL9 peptide can be the β-catenin interacting face.The “interacting face” of the alpha helix includes those amino acidresidues which interact with other amino acid residues on other proteinsand/or in other helices. Methods for identifying repeats and theinteracting face residues are well known in the art and describedherein.

As used herein, the term “hydrocarbon stapling”, refers to a process forstably cross-linking a polypeptide having at least two amino acids thathelps to conformationally bestow the native secondary structure of thatpolypeptide. Hydrocarbon stapling promotes or maintains a helicalsecondary structure in a peptide predisposed to have a helical secondarystructure, e.g., alpha-helical secondary structure, to attain ormaintain its native alpha-helical conformation. This secondary structureincreases resistance of the polypeptide to proteolytic cleavage andheat, and also may increase hydrophobicity.

The hydrocarbon stapled polypeptides include one or more tethers(linkages) between two non-natural amino acids (or a non-native linkage,e.g., carbon-carbon, from two natural amino acids), which tethersignificantly enhances the helical secondary structure of thepolypeptide. Generally, to promote a helical structure, the tetherextends across the length of one or two helical turns (i.e., about 3, 4,or 7 amino acids). Accordingly, amino acids positioned at i and i+3; iand i+4; or i and i+7 are ideal candidates for chemical modification andcross-linking. Thus, for example, where a peptide has the sequence . . .X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . , and the amino acid X isindependently selected for each position, cross-links between X1 and X4,or between X1 and X5, or between X1 and X8 are useful as are cross-linksbetween X2 and X5, or between X2 and X6, or between X2 and X9, etc. Theuse of multiple cross-links (e.g., 2, 3, 4 or more) is alsocontemplated. The use of multiple cross-links is effective atstabilizing and optimizing the peptide, especially with increasingpeptide length. The use of “stitched” cross-links has also been achievedwhereby double linkages are made from a common origin (e.g., X1, X5, andX9, where X5 is the anchor point for both staples). Thus, the inventionencompasses the incorporation of one or more crosslinks within thepolypeptide sequence. The use of multiple cross-links is effective atstabilizing and optimizing the peptide, especially with increasingpeptide length. Thus, the invention encompasses the incorporation of oneor more crosslinks within a polypeptide sequence, including stitchedcrosslinks in which two staples arise from a common origin.

As used herein, the term “staple scan” refers to the synthesis of alibrary of stapled peptides whereby the location of the i and i+3; i andi+4; and i and i+7 single and multiple staple, or stitches, arepositioned sequentially down the length of the peptide sequence,sampling all possible positions, to identify desired or optimalproperties and activities for the stapled or stitched constructs.

As used herein, the terms “identity” or “percent identity”, refers tothe subunit sequence similarity between two polymeric molecules, e.g.,two polynucleotides or two polypeptides. When a subunit position in bothof the two molecules is occupied by the same monomeric subunit, e.g., ifa position in each of two peptides is occupied by serine, then they areidentical at that position. The identity between two sequences is adirect function of the number of matching or identical positions, e.g.,if half (e.g., 5 positions in a polymer 10 subunits in length), of thepositions in two peptide or compound sequences are identical, then thetwo sequences are 50% identical; if 90% of the positions, e.g., 9 of 10are matched, the two sequences share 90% sequence identity. The identitybetween two sequences is a direct function of the number of matching oridentical positions. Thus, if a portion of the reference sequence isdeleted in a particular peptide, that deleted section is not counted forpurposes of calculating sequence identity. Identity is often measuredusing sequence analysis software e.g., BLASTN or BLASTP (available at(www.ncbi.nih.gov/BLAST). The default parameters for comparing twosequences (e.g., “Blast”-ing two sequences against each other), byBLASTN (for nucleotide sequences) are reward for match=1, penalty formismatch=−2, open gap=5, extension gap=2. When using BLASTP for proteinsequences, the default parameters are reward for match=0, penalty formismatch=0, open gap=11, and extension gap=1. Additional, computerprograms for determining identity are known in the art.

As used herein, “isolated” or “purified” when used in reference to apolypeptide means that a natural polypeptide or protein has been removedfrom its normal physiological environment (e.g., protein isolated fromplasma or tissue) or is synthesized in a non-natural environment (e.g.,artificially synthesized in an in vitro translation system or usingchemical synthesis). Thus, an “isolated” or “purified” polypeptide canbe in a cell-free solution or placed in a different cellular environment(e.g., expressed in a heterologous cell type). The term “purified” doesnot imply that the polypeptide is the only polypeptide present, but thatit is essentially free (about 90-95%, up to 99-100% pure) of cellular ororganismal material naturally associated with it, and thus isdistinguished from naturally occurring polypeptide. Similarly, anisolated nucleic acid is removed from its normal physiologicalenvironment. “Isolated” when used in reference to a cell means the cellis in culture (i.e., not in an animal), either cell culture or organculture, of a primary cell or cell line. Cells can be isolated from anormal animal, a transgenic animal, an animal having spontaneouslyoccurring genetic changes, and/or an animal having a genetic and/orinduced disease or condition.

As used herein, “kits” are understood to contain at least onenon-standard laboratory reagent for use in the methods of the invention.For example, a kit can include at least one of, preferably at least twoof at least one peptide, and instructions for use, all in appropriatepackaging. The kit can further include any other components required topractice the method of the invention, as dry powders, concentratedsolutions, or ready to use solutions. In some embodiments, the kitcomprises one or more containers that contain reagents for use in themethods of the invention; such containers can be boxes, ampules,bottles, vials, tubes, bags, pouches, blister-packs, or other suitablecontainer forms known in the art. Such containers can be made ofplastic, glass, laminated paper, metal foil, or other materials suitablefor holding reagents.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide without abolishing orsubstantially altering its activity/secondary structure (alpha-helicalstructure).

“Obtaining” is understood herein as manufacturing, purchasing, orotherwise coming into possession of.

As used herein, “operably linked” is understood as joined, preferably bya covalent linkage, e.g., joining an amino-terminus of one peptide to acarboxy terminus of another peptide, in a manner that the two or morecomponents that are operably linked either retain their originalactivity, or gain an activity upon joining such that the activity of theoperably linked portions can be assayed and have detectable activityusing at least one of the methods provided in the examples.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. For example,pharmaceutically acceptable carriers for administration of cellstypically is a carrier acceptable for delivery by injection, and do notinclude agents such as detergents or other compounds that could damagethe cells to be delivered. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, intramuscular,intraperitoneal, rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound that produces a therapeutic effect.

As used herein, “plurality” is understood to mean more than one. Forexample, a plurality refers to at least two, three, four, five, or more.

A “polypeptide” or “peptide” as used herein is understood as two or moreindependently selected natural or non-natural amino acids joined by acovalent bond (e.g., a peptide bond). A peptide can include 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturalor non-natural amino acids joined by peptide bonds. Polypeptides asdescribed herein include full length proteins (e.g., fully processedproteins) as well as shorter amino acids sequences (e.g., fragments ofnaturally occurring proteins or synthetic polypeptide fragments).

A “sample” as used herein refers to a biological material that isisolated from its environment (e.g., blood or tissue from an animal,cells, or conditioned media from tissue culture) and is suspected ofcontaining, or known to contain an analyte, such as a virus, anantibody, or a product from a reporter construct. A sample can also be apartially purified fraction of a tissue or bodily fluid. A referencesample can be a “normal” sample, from a donor not having the disease orcondition fluid, or from a normal tissue in a subject having the diseaseor condition (e.g., non-infected tissue vs. a infected tissue). Areference sample can also be from an untreated donor or cell culture nottreated with an active agent (e.g., no treatment or administration ofvehicle only). A reference sample can also be taken at a “zero timepoint” prior to contacting the cell or subject with the agent to betested.

“Similarity” or “percent similarity” in the context of two or morepolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residues,or conservative substitutions thereof, that are the same when comparedand aligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms, or by visual inspection.

The term “stable” or “stabilized”, as used herein with reference to apolypeptide, refers to polypeptides which have been hydrocarbon-stapledto promote and/or maintain helical structure and/or improve proteaseresistance and/or improve acid stability and/or improve thermalstability and/or improve pharmacologic properties. Stabilizedpolypeptides are a type of structurally constrained polypeptides.

As used herein, “structurally constrained peptides” and the like areunderstood to include modified peptides having any (i.e., at least one)chemical modification, e.g., mutation of the original or native sequencewith a natural or non-natural amino acid; chemical modification toincorporate a molecular tether; chemical modification to promote theformation of a disulfide bridge; etc. such that the structurallyconstrained peptide adopts a more limited number of structures than theunmodified peptide. A structurally constrained peptide can include 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more mutations as compared to thenative, wild-type sequence. For example, molecular tethers can includehydrocarbon staples to promote the formation of stable helicalstructures, especially alpha-helical and 3₁₀ structures, or kinksdepending on the positions of the ends of the tethers and the lengths ofthe tethers. Natural or non-natural amino acids can be employed topromote kinks (e.g., bends in the structure as defined by the variableangles between the two adjoining structures) or other preferredconfirmations. For example, the natural amino acid proline can induce akink in a peptide due to the structure of the amino acid R group and thelack of a hydrogen-bond donor. Non-natural amino acids, particularlythose having large and/or charged R groups, or N-methylated amides,N-substituted glycines, cyclic alpha,alpha-disubstitution, cyclicN,N-disubstitution, and beta-amino acids can promote specific, desiredconfirmations. It is understood that a population of “structurallyconstrained” peptides in solution may not all have the desiredconfirmation all of the time. Instead, in a population of structurallyconstrained peptides in solution, the desired confirmation is present atleast about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more of the timethan the native or original peptide sequence in solution prior tochemical modification. The structure of a population of peptides insolution can be determined by various methods known to those of skill inthe art including, but not limited to, circular dichroism and NMRspectroscopy. X-ray crystallography can be applied to determine thestructure of a constrained peptide when packed in the form of a crystal.

“Small molecule” as used herein is understood as a compound, typicallyan organic compound, having a molecular weight of no more than about1500 Da, 1000 Da, 750 Da, or 500 Da. In an embodiment, a small moleculedoes not include a polypeptide or nucleic acid including only naturalamino acids and/or nucleotides.

An agent, polypeptide, nucleic acid, or other compound “specificallybinds” a target molecule, e.g., antigen, polypeptide, nucleic acid, orother compound, when the target molecule is bound with at least100-fold, preferably at least 500-fold, preferably at least 1000-fold,preferably at least a 5000-fold, preferably at least a 10,000-foldpreference as compared to a non-specific compounds, or a pool ofnon-specific compounds. Specifically binds can be used in relation tobinding one of two or more related compounds that have physicallyrelated structures. Binding preferences and affinities, absolute orrelative, can be determined, for example by determining the affinity foreach pair separately or by the use of competition assays or othermethods well known to those of skill in the art.

A “subject” as used herein refers to living organisms. In certainembodiments, the living organism is an animal. In certain preferredembodiments, the subject is a mammal. In certain embodiments, thesubject is a domesticated mammal. Examples of subjects include humans,monkeys, dogs, cats, mice, rats, cows, horses, goats, and sheep. A humansubject may also be referred to as a patient.

A subject “suffering from or suspected of suffering from” a specificdisease, condition, or syndrome has a sufficient number of risk factorsor presents with a sufficient number or combination of signs or symptomsof the disease, condition, or syndrome such that a competent individualwould diagnose or suspect that the subject was suffering from thedisease, condition, or syndrome.

“Therapeutically effective amount,” as used herein refers to an amountof an agent which is effective, upon single or multiple doseadministration to the cell or subject, in prolonging the survivabilityof the patient with such a disorder, reducing one or more signs orsymptoms of the disorder, preventing or delaying infection, preventingor delaying the progression of a disease or disorder and the like beyondthat expected in the absence of such treatment.

An agent can be administered to a subject, either alone or incombination with one or more therapeutic agents, as a pharmaceuticalcomposition in mixture with conventional excipient, e.g.,pharmaceutically acceptable carrier, or therapeutic treatments.

The pharmaceutical agents may be conveniently administered in unitdosage form and may be prepared by any of the methods well known in thepharmaceutical arts, e.g., as described in Remington's PharmaceuticalSciences (Mack Pub. Co., Easton, Pa., 1985). Formulations for parenteraladministration may contain as common excipients such as sterile water orsaline, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. In particular,biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be usefulexcipients to control the release of certain agents.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to e.g., thespecific compound being utilized, the particular composition formulated,the mode of administration and characteristics of the subject, e.g., thespecies, sex, weight, general health and age of the subject. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

As used herein, “susceptible to” or “prone to” or “predisposed to” aspecific disease or condition and the like refers to an individual whobased on genetic, environmental, health, and/or other risk factors ismore likely to develop a disease or condition than the generalpopulation. An increase in likelihood of developing a disease may be anincrease of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₀ indicates that the group may have from 1 to10 (inclusive) carbon atoms in it. In the absence of any numericaldesignation, “alkyl” is a chain (straight or branched) having 1 to 20(inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20) carbon atoms in it. The term “alkylene” refers to adivalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkenyl” refers to aC₂-C₈ alkenyl chain. In the absence of any numerical designation,“alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkynyl” refers to aC₂-C₈ alkynyl chain. In the absence of any numerical designation,“alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Preferred cycloalkyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. This includes all individual sequences when arange of SEQ ID NOs: is provided. For example, a range of 1 to 50 isunderstood to include any number, combination of numbers, or sub-rangefrom the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

The symbol

when used as part of a molecular structure refers to a single bond or atrans or cis double bond.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Pharmaceutical Compositions and Routes of Administration

One or more structurally constrained peptide of the instant inventioncan be used in a pharmaceutical composition for the treatment of adisorder provided herein. Treatment method provided herein can beperformed using a combination of the structurally constrained peptides,which can be selected and combined to treat the disorder in the subject.For example, a pharmaceutical composition of the instant invention caninclude 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or morestructurally constrained peptides. The structurally constrained peptidescan also be combined with other agents, e.g., anti-cancer agents orangiogenesis inhibitors.

As used herein, the compounds of this invention are defined to includepharmaceutically acceptable derivatives thereof. A “pharmaceuticallyacceptable derivative” means any pharmaceutically acceptable salt,ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives are those that increase thebioavailability of the compounds of this invention when such compoundsare administered to a mammal (e.g., by allowing an orally administeredcompound to be more readily absorbed into the blood, to increase serumstability or decrease clearance rate of the compound) or which enhancedelivery of the parent compound to a biological compartment (e.g., thebrain or lymphatic system) relative to the parent species. Derivativesinclude derivatives where a group which enhances aqueous solubility oractive transport through the gut membrane is appended to the structureof formulae described herein.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion. Pharmaceutically acceptablesalts of the compounds of this invention include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acid salts include acetate, adipate, benzoate,benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate,formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,phosphate, picrate, pivalate, propionate, salicylate, succinate,sulfate, tartrate, tosylate and undecanoate. Salts derived fromappropriate bases include alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N-(alkyl)₄₊ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersible products may be obtained by such quaternization.

The compounds of the invention can, for example, be administered byinjection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, intravaginally, cervically,topically, in an ophthalmic preparation, or by inhalation, with a dosageranging from about 0.001 to about 100 mg/kg of body weight, or accordingto the requirements of the particular drug and more preferably from0.5-10 mg/kg of body weight. The methods herein contemplateadministration of an effective amount of compound or compoundcomposition to achieve the desired or stated effect.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain from about 1% to about 95% active compound(w/w). Alternatively, such preparations contain from about 20% to about80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

Pharmaceutical compositions of this invention comprise compounds of theinvention or a pharmaceutically acceptable salt thereof; an additionalagent including for example, one or more therapeutic agents for theprevention and/or treatment of a disorder provided herein, particularlyfor the prevention and/or treatment of cancer, and any pharmaceuticallyacceptable carrier, adjuvant or vehicle. Alternate compositions of thisinvention comprise a compound of the invention or a pharmaceuticallyacceptable salt thereof; and a pharmaceutically acceptable carrier,adjuvant or vehicle. The compositions delineated herein include thecompounds of the invention delineated herein, as well as additionaltherapeutic agents if present, in amounts effective for achieving amodulation of disease or disease symptoms, including cancer or symptomsthereof.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α.-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tween® or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropyle-ne-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as alpha-, beta-, and gamma-cyclodextrin,may also be advantageously used to enhance delivery of compounds of theformulae described herein.

The pharmaceutical compositions of this invention may be administeredenterally for example by oral administration, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir, preferably by oral or vaginal administrationor administration by injection. The pharmaceutical compositions of thisinvention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases, or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, and intracranial injection orinfusion techniques.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a patient,including suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween® 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and/or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase and is combined with emulsifying and/or suspendingagents. If desired, certain sweetening and/or flavoring and/or coloringagents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

The pharmaceutical compositions of the invention may be administeredtopically or intravaginally. The pharmaceutical composition will beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. In still another embodiment, the pharmaceuticalcomposition is formulated as a vaginal ring. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water. The pharmaceutical compositions of this invention may also betopically applied to the lower intestinal tract by rectal suppositoryformulation or in a suitable enema formulation. Topically-transdermalpatches and iontophoretic administration are also included in thisinvention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

Effective dosages of the peptides of the invention to be administeredmay be determined through procedures well known to those in the artwhich address such parameters as biological half-life, bioavailability,and toxicity.

A therapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in humans. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (e.g., theconcentration of the test compound which achieves a half-maximalinhibition of the BCL9/b-catenin protein interaction or functionalsurrogate thereof as measured by an assay relative to the amount of theevent in the absence of the test compound) as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography (HPLC) or mass spectrometry(MS).

Kits

The present invention also encompasses a finished packaged and labeledpharmaceutical product or laboratory reagent. This article ofmanufacture includes the appropriate instructions for use in anappropriate vessel or container such as a glass vial or other containerthat is hermetically sealed. A pharmaceutical product may contain, forexample, a compound of the invention in a unit dosage form in a firstcontainer, and in a second container, sterile water or adjuvant forinjection. Alternatively, the unit dosage form may be a solid suitablefor oral, transdermal, intranasal, intravaginal, cervical ring, ortopical delivery.

In a specific embodiment, the unit dosage form is suitable forintravenous, intramuscular, intraperitoneal, intranasal, oral,intravaginal, cervical, topical or subcutaneous delivery. Thus, theinvention encompasses solutions, solids, foams, gels, preferablysterile, suitable for each delivery route.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician, or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instructions indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures (e.g., detection and quantitation of infection), and othermonitoring information.

Specifically, the invention provides an article of manufacture includingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of a pharmaceutical agent contained withinsaid packaging material, wherein said pharmaceutical agent comprises acompound of the invention, and wherein said packaging material includesinstruction means which indicate that said compound can be used tomanage, treat, and/or ameliorate one or more symptoms associated with adisease provided herein, by administering specific doses and usingspecific dosing regimens as described herein.

The following examples are provided merely as illustrative of variousaspects of the invention and shall not be construed to limit theinvention in any way.

Disorders Treated by the Invention

In certain embodiments, the disease or disorder treated by thestabilized peptides of the invention is associated with angiogenesis. Incertain embodiments, the disease is selected from: tumor or cancergrowth (neoplasia), skin disorders, neovascularization, inflammatory andarthritic diseases, retinoblastoma, cystoid macular edema (CME),exudative age-related macular degeneration (AMD), diabetic retinopathy,diabetic macular edema, or ocular inflammatory disorders.

In various embodiments, the structurally constrained peptides of theinvention can be used for overcoming cancer stem cell chemo- andradioresistance (treatment-resistance).

In certain embodiments, the disease or disorder is tumor or cancergrowth (neoplasia). In a further embodiment, the disease or disorder isocular cancer, rectal cancer, colon cancer, colorectal caner, cervicalcancer, prostate cancer, breast cancer, bladder cancer, oral cancer,benign and malignant tumors, stomach cancer, liver cancer, pancreaticcancer, lung cancer, corpus uteri, ovary cancer, prostate cancer,testicular cancer, renal cancer, brain/cns cancer, throat cancer,multiple myeloma, skin melanoma, acute lymphocytic leukemia, acutemyelogenous leukemia, Ewing's Sarcoma, Kaposi's Sarcoma, basal cellcarcinoma and squamous cell carcinoma, small cell lung cancer,choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma,Wilms Tumor, neuroblastoma, mouth/pharynx cancer, esophageal cancer,larynx cancer, lymphoma, neurofibromatosis, tuberous sclerosis,hemangiomas, and lymphangiogenesis.

In other embodiments, the disease or disorder is a skin disorder. In afurther embodiment, the disease or disorder is psoriasis, acne, rosacea,warts, eczema, hemangiomas, lymphangiogenesis, Sturge-Weber syndrome,venous ulcers of the skin, neurofibromatosis, and tuberous sclerosis.

In certain embodiments, the disease or disorder is neovascularization.In a further embodiment, the disease or disorder is diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma, retrolental fibroplasias, epidemickeratoconjunctivitis, vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,Sjogren's, acne rosacea, phlyctenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, herpes simplex infections, herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginaldegeneration, marginal keratolysis, trauma, rheumatoid arthritis,systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis,Stevens-Johnson disease, pemphigoid, radial keratotomy, corneal graftrejection, macular edema, macular degeneration, sickle cell anemia,sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, veinocclusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme disease, systemic lupuserythematosus, retinopathy of prematurity, Eales' disease, Behcet'sdisease, infections causing a retinitis or choroiditis, presumed ocularhistoplasmosis, Best's disease, myopia, optic pits, Stargardt's disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications, and diseasesassociated with rubeosis (neovascularization of the ankle).

In certain embodiments, the disease or disorder is inflammatory andarthritic disease. In a further embodiment, the disease or disorder isrheumatoid arthritis, osteoarthritis, lupus, scleroderma, Crohn'sdisease, ulcerative colitis, psoriasis, sarcoidosis, Sarcoidosis, skinlesions, hemangiomas, Osler-Weber-Rendu disease, hereditary hemorrhagictelangiectasia, and osteoarthritis.

In other embodiments, the disease or disorder affects the dermis,epidermis, endometrium, retina, surgical wound, gastrointestinal tract,umbilical cord, liver, kidney, reproductive system, lymphoid system,central nervous system, breast tissue, urinary tract, circulatorysystem, bone, muscle, or respiratory tract.

EXAMPLES Example 1. Peptide Synthesis and Circular Dichroism

To generate stabilized alpha-helices of the BCL9 HD2 domain, whichdirectly interacts with b-catenin (FIG. 16), syntheses of hydrocarbonstapled peptides (FIG. 14, FIG. 15) were performed as previouslydescribed (Walensky, L. D. et al. Science 305, 1466-70 (2004); Bird, G.H., et al., Methods Enzymol 446, 369-86 (2008); Bird et al. PNAS 107,14093-8, (2010)). Peptides were produced on an Apex 396 (Aapptec)automated peptide synthesizer using Rink amide AM LL resin (EMDBiosciences, 0.2 mmol/g resin), at 50 mmol scale. The standard Fmocprotocol employed 2×10 min deprotections in 20% piperidine/NMP followedby a pair of consecutive methanol and dimethylformamide (DMF) washes.The incorporated non-natural amino acids were treated with 4×10 minincubations in 20% piperidine/NMP to achieve complete deprotection.Amino acid coupling was performed using 0.4 M stock solutions ofFmoc-protected amino acids, 0.67 M2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and 2 M N,N-diisopropyl ethylamine (DIEA),yielding 1 mL of 0.2 M active ester (4 equivalents). Coupling frequencyand incubation times were 2×30 min for standard residues, 2×45 min forthe olefinic non-natural amino acids, and 3×45 min for the residuefollowing a non-natural amino acid. Upon completion of automatedsynthesis, the amino terminus was either acetylated or capped withFmoc-β-Ala for FITC derivatization. To generate hydrocarbon staples byolefin metathesis, the resin was charged with a 10 mM solution ofbis(tricyclohexylphosphine)-benzylidene ruthenium (IV) dichloride(Grubbs' first generation catalyst) in 1,2-dichloroethane and stirredfor 2 hours twice. For FITC derivatization, Fmoc-β-Ala was deprotectedwith piperidine in NMP and then reacted with fluorescein isothiocyanate(FITC) and triethylamine in dimethylformamide overnight. The peptide wascleaved from the resin and deprotected in TFA/triisopropyl silane(TIS)/water (95%, 2.5%, 2.5%), and precipitated withdiethylether/hexanes. Stapled peptides were purified by reverse-phaseHPLC (Agilent) using a C18 column (Zorbax), characterized by LC/MS (massspectra obtained using electrospray in positive ion mode), andquantified by amino acid analysis (AAA) on a Beckman 6300high-performance amino acid analyzer. Working stock solutions weregenerated by dissolving the lyophilized powder in 100% DMSO at 1 to 10mM. SAH-gp41 powder and DMSO solutions were stored at −20° C.Determination of α-helicity was performed as previously described(Walensky, L. D. et al. Science 305, 1466-70 (2004); Bird, G. H., etal., Methods Enzymol 446, 369-86 (2008)). See experimental results inFIG. 1A-FIG. 1C, FIG. 1G.

Example 2. Protein Production and Purification

Recombinant human BCL9 (214-493) was cloned into pET-23a (+) was clonedinto pET-23a (+) vector (Novagen) containing carboxy-terminalhexa-histidine tag (SEQ ID NO: 141) E. coli BL21 (DE3) competent cells(Stratagene) were transformed, incubated at 37° C. until A600=0.6 wasreached and then induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG)for 3 h. Cells were harvested by centrifugation and lysed by sonicationin 50 mM Na₂HPO₄, pH 8.0, 0.3M NaCl buffer. The lysates were thencentrifuged and loaded onto HIS-Select Nickel Affinity Gel (Sigma) andwashed with wash buffer (50 mM NaH₂PO₄, pH 8.0, 0.3M NaCl and 10 mMimidazole). The protein was eluted in 50 mM Na₂HPO₄, pH 8.0, 0.3M NaCland 250 mM imidazole and dialyzed overnight in sterile 1×PBS. Humanβ-catenin constructs (e.g., residues 1-781, 138-683, 273-684) werecloned into pGEX4T1/pGEX4T2, pET-28a, and pET-23a respectively.His-tagged fusion proteins were generated as described above for BCL9.For GST-tagged constructs, transformed E. coli BL21 (DE3) were culturedat 37° C. to A600=0.6 and induced with 1 mM IPTG for 4 h. Cells werepelleted, resuspended and sonicated in Buffer A (50 mM Tris, pH 8.0, 150MM NaCl, sucrose 20%, 5 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM PMSF, 2mg/ml aprotinin, and 0.7 mg/ml pepstatin). Solubilized proteins wereadsorbed to glutathione-Sepharose 4B beads (GE), which were then elutedin Buffer A with 20 mM glutathione and dialyzed against PBS buffersupplemented with protease inhibitor cocktail tablets (Roche).

Example 3. GST Pull-Down Assays

Equal amounts (0.5 μM) of His-tagged BCL9 and GST-tagged β-catenin boundto glutathione-Sepharose 4B beads (GE) were incubated with or withoutincreasing amounts of HD2 or SAH-BCL9 peptides for 1 h at 4° C. in afinal volume of 1000 μl PBS. Protein complexes were pelleted bycentrifugation at 2000 rpm for 2 min and beads washed four times withPBS buffer. The beads were then taken up in SDS-PAGE loading buffer,boiled, and SDS-PAGE performed to visualize bound proteins by Coomassiestaining.

Example 4. Patient Samples and Cell Lines

Bone marrow specimens were obtained from patients with MM in accordancewith Dana-Farber Cancer Institute Review Board approval and informedconsent performed in compliance with the Declaration of Helsinki.Primary CD138+ plasma cells were purified using magnetic beads asdescribed (Sukhdeo, K. et al. Proc Natl Acad Sci USA 104, 7516-21(2007)). CRC primary tumor samples were obtained from the Brigham andWomen's Hospital in accordance with the policies of their InstitutionalReview Board. To generate sufficient CRC primary tumor cells forexperimentation, the primary tumors were first expanded subcutaneouslyin NOD/SCID mice (Jackson Laboratory). After the tumors reached 2 cm indiameter, mice were sacrificed according to institutional guidelines andsubcutaneous tumor xenografts were minced with a scalpel and digested byincubation with collagenase IV (Worthington Biomedical Corporation) and0.01% DNase I (Sigma-Aldrich) at 37° C. for 30 min, followed byadditional mechanical disaggregation using a Stomacher device (SewardLaboratory Systems Inc.). Samples were filtered through a 70 μm cellstrainer and washed with PBS. Red blood cells were lysed using ACKlysing buffer (BioWhittaker, Lonza) and viable tumor cells were enrichedby Ficoll-Paque gradient centrifugation (GE Healthcare). To purifyviable tumor cells only, the samples were treated with APC conjugatedanti-mouse H-2Kd (clone SF1-1.1.1, eBioscience), FITC-conjugatedanti-human EpCAM antibodies (clone Ber-EP4, Dako), and Hoechst 33258(Sigma-Aldrich), and then FACSAria flow sorting (BD Biosciences) wasused to isolate the EpCAM-positive, H-2Kd-negative, and Hoechst-negativeprimary tumor cells. Cultured cell lines were maintained as previouslydescribed (Mani, M. et al. Cancer Res 69, 7577-86 (2009)). See resultsin FIG. 3B, FIG. 3D.

Example 5. Immunoblotting and Co-Immunoprecipitation

Western blotting, performed as described (Sukhdeo, K. et al. Proc NatlAcad Sci USA 104, 7516-21 (2007)), employed the following primaryantibodies: BCL9 (6109) (Mani, M. et al. Cancer Res 69, 7577-86 (2009)),BCL9 (ab37305, Abcam), B9L (AF4967, R&D Systems), β-catenin (CATS-H10,Zymed), FITC (ab19224, Abcam), Actin-HRP (C-11, Santa Cruz), Caspase3(#9662, Cell Signaling), IxBα (#9242, Cell signaling), PARP (#9542, CellSignaling), E-cadherin (#3195, Cell Signaling), and Lamin B (sc-6217,Santa Cruz). Horseradish peroxidase conjugated secondary antibodies werepurchased from Santa Cruz and SouthernBiotech. Co-immunoprecipitationwas performed as described (Walensky, L. D. et al. Science 305, 1466-70(2004)). Briefly, cells were lysed in 50 mM Tris, 150 mM NaCl, and 1%CHAPS buffer containing protease and phosphatase inhibitors. Lysateswere precleared with Protein A/G PLUS-agarose beads (Santa CruzBiotechnologies) for 3 hours followed by overnight incubation at 4° C.with the respective antibodies. Agarose A/G beads were then added for 4h, pelleted, and washed as described (Walensky, L. D. et al. Science305, 1466-70 (2004)). See results in FIG. 1D, FIG. 1H, FIG. 2A, FIG. 8.

Example 6. SAH-BCL9 Cellular Uptake and Localization Analyses

For fluorescence microscopy evaluation, cells were prepared using acytocentrifuge (Thermo Shandon) and fixed as previously described(Sukhdeo, K. et al. Proc Natl Acad Sci USA 104, 7516-21 (2007); Mani, M.et al. Cancer Res 69, 7577-86 (2009)). Anti-β-catenin andrhodamine-conjugated secondary antibodies (5 μg/ml; SouthernBiotechnology) were employed. Images were obtained using a BioRadRadiance 2000 laser scanning confocal microscope. Cell permeability ofSAH-BCL9_(B) and SAH-BCL9_(B)(R359E) were determined by fluorescencemicroscopy of cells treated with FITC-derivatives of the above describedstapled peptides and also by blotting/fluorescence scan, performed aspreviously described (Fitter, K. et al Methods Enzymol, 446, (2008),387-408; Walensky, L. D. et al. Science 305, 1466-70 (2004). See resultsin FIG. 1E, FIG. 7.

Example 7. Histopathological Analysis and Immunohistochemistry

Tissue sections were process as described (Sukhdeo, K. et al. Proc NatlAcad Sci USA 104, 7516-21 (2007)). Sections were incubated with primaryantibodies (5 μg/ml) or the corresponding IgG fraction of preimmuneserum overnight at 4° C. in blocking solution (3% BSA/PBS). BCL9(ab37305, Abcam), mouse CD34 (RAM34, eBiosciences), human CD34 (M7165,Dako) and human CD44H (2C5, R&D Systems) antibodies were employed. Bloodvessel formation in the CRC and MM models was evaluated using anti-mouseCD34 and anti-human CD34 antibodies, respectively, and the correspondingbiotinylated antibodies coupled to streptavidin peroxidase (Vector). Thenumber of blood vessels was determined by counting the mean number ofindependent blood vessels in 5 randomly selected fields at 50×magnification as highlighted by CD34 staining (brown color). See resultsin FIG. 4B, FIG. 4E, FIG. 4I, FIG. 6.

Example 8. Quantitative Reverse Transcription-PCR

RNA was extracted with TRIzol Reagent (Invitrogen) according to themanufacturer's protocol. Total RNA (2 μg) was reverse transcribed(SuperScript VILO cDNA synthesis kit, Invitrogen) and qPCR was performedusing an Applied Biosynthesis 7500 Real-time PCR system. Analysis oftarget genes was conducted in quadruplicate using POWER SYBR GreenMaster Mix (Applied Biosystems) with previously described primer sets.Transcripts levels were normalized to β-actin expression. Theseexperiments were repeated three times. See results in FIG. 2B, FIG. 2C.

Example 9. Gene Expression Profiling

RNA from SAH-BCL9_(B) and vehicle (0.1% DMSO)-treated cells were run onan Affymetrix U133A 2.0 array chip as described (Mollering et al, Nature462, 182-8 (2009). Statistical analyses were performed in R(http://www.r-project.org). The array data were normalized with rmamethod (Bolstad, B. M., et al. Bioinformatics 19, 185-93 (2003)) asimplemented in the Affy package(http://www.bioconductor.org/packages/2.6/bioc/html/affy.html) anddifferential expression calculated with empirical Bayes shrinkage of thestandard errors toward a common value with LIMMA(http://www.bioconductor.org/packages/2.6/bioc/html/limma.html)(McCarthy, D. J. & Smyth, G. K. Bioinformatics 25, 765-71 (2009); Smyth,G. K. Stat Appl Genet Mol Biol 3, Article3 (2004)). Gene set enrichmentanalysis was performed using GSEA software (version 2.06) and mSigDB(version 2.5) (Subramanian, A. et al. Proc Natl Acad Sci USA 102,15545-50 (2005)).

Example 10. Cell Proliferation, Viability Assay and Detection ofApoptosis

Cell proliferation assays were performed as described (Sukhdeo, K. etal. Proc Natl Acad Sci USA 104, 7516-21 (2007)). Cell viability wasmeasured using the CellTiter-Glo assay (Promega) according to themanufacturer's instructions. Apoptosis was evaluated by activatedcaspase-3 and PARP western blotting. See results in FIG. 3A-FIG. 3F,FIG. 13, and FIG. 20.

Example 11. Angiogenesis and Invasion Assays

Angiogenesis was evaluated as previously described (Mani, M. et al.Cancer Res 69, 7577-86 (2009)) using an in vitro angiogenesis assay kit(Millipore). For capillary tube formation analysis, HUVEC were culturedon polymerized matrix gel and exposed to supernatant media collectedfrom Colo320 or MM1S cells treated with vehicle (0.5% DMSO),SAH-BCL9_(B) peptides (5 μM) for 24 h. The number of capillary tubesformed after 5 h treatment at 37° C. was determined by counting 5randomly selected fields at 40× magnification, according tomanufacture's instructions. HUVEC cultured in VEGF media and VEGF-freemedia were used as positive and negative controls, respectively.Cellular invasion assays were performed using Matrigel Boyden chambers(BD Biosciences) as described (Mani, M. et al. Cancer Res 69, 7577-86(2009)). The reported data represent the average of three independentexperiments performed in triplicate. See results in FIG. 3H-FIG. 3I.

Example 12. In Vivo Anti-Tumor Effect of SAH-BCL9_(B) (Xenograft Models)

GFP-positive Colo320 cells were generated as previously reported (Mani,M. et al. Cancer Res 69, 7577-86 (2009)). Cells were pelleted,resuspended in sterile 1×PBS and injected intraperitoneally (1×10⁶cells/mouse) into 5-week-old sublethally irradiated NOD.CB17-PrkdcSCID/Jmice (Jackson Laboratory) (n=6 per cohort). Two days after cellularinoculation, mice were treated by intraperitoneal injection with vehicle(2.5% DMSO in D5W) or SAH-BCL9 peptides (20 mg/kg) on alternate days fora total of 6 doses. Forty days after tumor cell injection, the mice wereeuthanized and GFP-positive tumor visualized using an ImageQuantLAS-4000 (GE Healthcare). Complete necropsies were performed for eachexperimental animal and livers were sectioned in their entirety at 5 mmintervals for quantitation of tumor metastases. Tissues were subjectedto H&E staining and immunohistochemical analysis using anti-CD34 andanti-CD44 antibodies.

For the SCID-hu murine model of human MM, human fetal bone graftsmeasuring 1.5×0.5 cm were subcutaneously implanted into eight week oldmale CB-17 SCID mice (Taconic) as previously described (Tassone, P. etal. Blood 106, 713-6 (2005)). Four weeks after bone implantation, 5×10⁶GFP-positive INA-6 MM cells were injected directly into each boneimplant. Two days later, mice were treated with 100 μl injections ofvehicle (2.5% DMSO in D5W) or SAH-BCL9 peptides (5 mg/kg) instilledadjacent to the bone chips on alternate days for a total of 10 doses.Mouse sera were serially monitored for shuIL-6R levels by ELISA (R&DSystems). Thirty-three days after tumor cell injection, the mice weresacrificed and analyzed for tumor burden by fluorescence imaging andhistologic analysis of the bone grafts. See results in FIG. 4A-FIG. 4I.In addition, TUNEL staining was performed on samples obtained from themice. The results demonstrated that there is an increase in apoptotictumor cells in animals treated with SAH-BCL9_(B) compared to vehicle orSAH-BCL9_(MUT)-treated mice. See results in FIG. 22 and FIG. 23. Allanimal experiments were performed in accordance with approved protocolsof the Dana-Farber Cancer Institute Animal Care and Use Committee.

Example 13. In Vivo Effect of SAH-BCL9_(B) on Wnt Reporter Activity

HCT116 cells were transfected with pOT-Luc plasmid or a control UbC-Lucplasmid. The HCT116-pOT-Luc cells were implanted into mice (n=2) on theleft flank and the constitutive UbC-Luc control cells on the rightflank. Animals underwent baseline imaging, followed by SAH-BCL9_(B) orSAH-BCL9_(B)(R359E) injection and serial imaging at the indicated timepoints. The pOT-Luc reporter activity was normalized to UbC-Lucactivity. See results in FIG. 9.

Example 14. VEGF ELISA

VEGF ELISA was performed as previously described (Mani, M. et al. CancerRes 69, 7577-86 (2009)). Briefly, cells (1×10⁶) were treated withvehicle and SAH-BCL9 peptides (5 μM) for 24 h. VEGF levels in thesupernatant were then measured according to the manufacturer's ELISAprotocol (DuoSet, R&D Systems).

Example 15. Chromatin Immunoprecipitation (ChIP) and Polymerase ChainReaction (PCR)

Antibody (3 μg) was prebound for 8 h to protein A and protein G Dynalmagnetic beads (Dynal Biotech, Norway) and washed 5 times with ice-coldPBS containing 5% BSA, and then added to the diluted chromatin forovernight immunoprecipitation using the following antibodies: TCF-4(Upstate #05-511), mouse IgG2a isotype control (Sigma, M5409), andrabbit IgG (sc-2027, Santa Cruz). The magnetic bead-chromatin complexeswere collected and washed 6× in RIPA buffer (50 mM HEPES [pH 7.6], 1 mMEDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5 M LiCl). DNA was eluted fromthe beads as previously described (Clevers, H. Cell 127, 469-80 (2006)).Amplification was carried out with a PTC-200 programmable thermalcontroller (MJ Research) after an initial denaturation at 94° C. for 5min, followed by 30 cycles of PCR using the following temperature andtime profile: denaturation at 94° C. for 0.5 min, primer annealing at59° C. for 0.5 min, primer extension at 72° C. for 0.5 min, and a finalextension of 72° C. for 10 min. The PCR products were visualized by 2%gel electrophoresis. The following promoter primer sets were employed:(1) VEGF: F (Forward): 5′-gcgtgtctctggacagagttt-3′ (SEQ ID NO: 116) andR (Reverse): 5′-agcctcagcccttccaca-3′(SEQ ID NO: 117); (2) VEGFupstream: F: 5′-gaggctatgccagctgtagg-3′(SEQ ID NO: 118) and R:5′-cccttttcctccaactctcc-3′(SEQ ID NO: 119); (3) c-Myc: F:5′-actcccccggctcggtccacaagc-3′, (SEQ ID NO: 120) and R:5′-cccaatttctcagccaggtttcag-3′ (SEQ ID NO: 121) (Klaus, A. & Birchmeier,W. Nat Rev Cancer 8, 387-98 (2008)). See results in FIG. 11A.

Example 16. VEGF Promoter Luciferase Assays

The VEGF promoter-driven luciferase constructs (2.6-kb) were a kind giftfrom Soumitro Pal (Transplantation Research Center, Children's HospitalBoston and Brigham and Women's Hospital) (Basu, A. et al. Cancer Res 68,5689-98 (2008)). Cells were transfected with the VEGF luciferaseconstructs using FuGENE transfection reagent (Roche) and luciferaseactivity was measured using Dual Luciferase Reporter Assay System(Promega) as previously described (Sukhdeo, K. et al. Proc Natl Acad SciUSA 104, 7516-21 (2007)). See results in FIG. 11B.

Example 17. Lentiviral Vectors

A lentiviral reporter vector containing seven TCF/LEF-1 binding motifsand a minimal promoter driving destabilized GFP expression (7×TdG) wasderived from the lentiviral vector TOP-dGFP, which contains threeTCF/LEF-1 binding motifs (Sukhdeo, K. et al. Proc Natl Acad Sci USA 104,7516-21 (2007)). Two synthetic complementary oligonucleotides (IDT-DNA)with four TCF/LEF-1 binding motifs (GATCAAAGG) were designed to generatecompatible overhanging ends for annealing to an Xba1 restriction site.The oligonucleotides were annealed by heating to 95° C. and slow coolingto room temperature, followed by ligating into the Xba1-linearizedTOP-dGFP vector, yielding 7×TdG. For construction of the control vectorcarrying seven FOP-sites (7×FdG), the 7×TOP cassette was removed fromthe 7×TdG vector by restriction digesting with Xma1 and Age1. Asynthetic cassette carrying seven FOP sites (GGCCAAAGG) but otherwiseidentical to the removed 7×TOP cassette was inserted, yielding 7×FdG.BCL9 shRNA and control shRNA lentiviral vectors were generated asreported (Sampietro, J. et al. Mol Cell 24, 293-300 (2006)). See resultsin FIG. 10, FIG. 11B.

Example 18. Lentivirus Production and Infection

HEK293T cells were plated in 10 cm tissue culture dishes andco-transfected with 10 μg lentiviral vector (either 7×TdG or 7×FdG), 10μg pCMV-dR8.91 and 2 μg pMD2.G (Naldini et al, PNAS, 1996) using 60 μLLipoD293 (Signagen) according to the manufacturer's protocol. The mediawas replaced after 12 h with 30% FCS containing DMEM (Gibco) andconditioned for 36 h. Conditioned medium was then filtered through 0.45μm syringe filters (Millipore), mixed 1:1 with fresh DMEM, and thendirectly used for infection of cultured Colo320 (ATCC) cells. Polybrene(Sigma) was added to a final concentration of 8 μg/mL to enhance theefficiency of infection. Lentivirus shRNA infections to knockdown BCL9expression were performed as described previously (Logan, C. Y. & Nusse,R. Annu Rev Cell Dev Biol 20, 781-810 (2004)). Briefly, recombinant BCL9shRNA and control lentiviruses were produced by transient transfectionof 293T cells. Colo320 were transduced with virus supernatant containingpolybrene, and GFP-expressing cells sorted by FACS. See results in FIG.10, FIG. 11B.

Example 19. Establishment of Single Cell Cultures

Colo320 cells were subjected to infection for 72 h with either 7×TdG or7×FdG lentivirus, and the transduced and control non-transduced cellswere trypsinized, washed, and then analyzed on a FACSaria flow sorter.Hoechst 33258 staining was used to exclude dead cells. SingleGFP-positive Colo320-7×TdG cells were sorted into 96 well plates usingstringent gating on forward/side scatter height and width to excludedoublets. The presence of a single cell per well was confirmedmicroscopically after sorting and then single cell cultures wereexpanded for subsequent use. See results in FIG. 11B.

Example 20. Chromatin Immunoprecipitation

Three micrograms of antibody was prebound for 8 h to protein A andprotein G Dynal magnetic beads (Dynal Biotech, Norway) and washed 5×with ice-cold PBS containing 5% BSA, and then added to the dilutedchromatin for overnight immunoprecipitation using the followingantibodies: TCF-4 (Upstate #05-511), mouse IgG2a isotype control (Sigma,M5409), and rabbit IgG (sc-2027, Santa Cruz). The magneticbead-chromatin complexes were collected and washed 6× in RIPA buffer (50mM HEPES [pH 7.6], 1 mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5 MLiCI). DNA was eluted from the beads as previously described (Shang, Y.,et al. Cell 103, 843-52 (2000)). Amplification was carried out with aPTC-200 programmable thermal controller (MJ Research) after an initialdenaturation at 94° C. for 5 min, followed by 30 cycles of PCR using thefollowing temperature and time profile: denaturation at 94° C. for 0.5min, primer annealing at 59° C. for 0.5 min, primer extension at 72° C.for 0.5 min, and a final extension of 72° C. for 10 min. The PCRproducts were visualized by 2% gel electrophoresis. The followingpromoter primer sets were employed: (1) VEGF^(B): F (Forward):5′-gcgtgtctctggacagagttt-3′ (SEQ ID NO: 116) and R (Reverse):5′-agcctcagcccttccaca-3′(SEQ ID NO: 117); (2) VEGF upstream: F:5′-gaggctatgccagctgtagg-3′(SEQ ID NO: 118) and R:5′-cccttttcctccaactctcc-3′ (SEQ ID NO: 119); (3) c-Myc: F:5′-actcccccggctcggtccacaagc-3′ (SEQ ID NO: 120), and R:5′-cccaatttctcagccaggtttcag-3′ (SEQ ID NO: 121). See results in FIG.11A.

Example 21. Reporter Assays

Luciferase activity was measured using the Dual Luciferase ReporterAssay System (Promega) as previously described (Sukhdeo, K. et al. ProcNatl Acad Sci USA 104, 7516-21 (2007)). To measure Wnt or NFκB reporteractivity, Colo320 cells were transfected with TOP-FLASH, FOP-FLASHplasmid (Millipore Corporation) or NFκB luciferase reporter(Stratagene), along with an internal Renilla control plasmid (hRL-null).Transfection was accomplished using FuGENE (Roche) according to themanufacturer's protocol. The results were normalized to control Renillaactivity. The reported data represent the average of three independenttransfection experiments performed in triplicate. See results in FIG. 9,FIG. 10.

Example 22. Selective Dissociation of the BCL9/β-Catenin Complex bySAH-BCL9_(B)

To evaluate binding by ELISA, glutathione microtiter plates (Pierce)were incubated with 50 ng recombinant GST-β-catenin in 100 μL of ELISAbuffer (PBS, 1% BSA, 0.05% Tween-20) per well and rotated (200 rpm) at37° C. for 1 hr, followed by 4-cycles of automated plate-washing withPBS, 0.05% Tween-20. Two-fold serial dilution of FITC-conjugatedpeptides in ELISA buffer were prepared in a separate 96-well plate andtransferred (100 μL) to the β-catenin-bound plate. The experimentalplate was incubated for 2 hr at 37° C. (200 rpm), subjected to automatedplate washing, and then 100 μL of a 1:7500 dilution ofanti-FITC-conjugated HRP in ELISA buffer was transferred to each wellfor an additional 1 hr incubation at 37° C. (200 rpm), followed byautomated plate washing. Wells were developed by adding 50 μL oftetramethylbenzidine (TMB) solution, incubating at room temperature for20 min, and then stopping the reaction with 50 μL of 2 M H2504. Theabsorbance at 450 nm was read on a microplate reader (Molecular Devices)and the binding isotherms plotted and EC50 values determined bynonlinear regression analysis using Prism software (GraphPad). Bindingassays were performed in triplicate and repeated at least twice withfreshly prepared recombinant proteins. Consistent with the reducedcapacity of FITC-SAH-BCL9_(MUT) (SAH-BCL9_(B)(R359E)) toimmunoprecipitate native β-catenin (see FIG. 1H), R359E pointmutagenesis caused a 5-fold decrease in direct binding activity torecombinant β-catenin protein. See results in FIG. 17A.

To evaluate the capacity of SAH-BCL9 to disrupt preformed BCL9/β-catenincomplexes, the biological activity required for Wnt signaling blockade,recombinant human BCL9 (residues 243-469) cloned into pET-23a (+) vectorcontaining carboxy-terminal hexa-histidine tag (SEQ ID NO: 141)(His-BCL9) and full-length human β-catenin cloned into pGEX-4T-1 vectorwith an amino-terminal glutathione-S-transferase (GST) tag (recombinantGST-β-catenin) were expressed and purified as previously reported (J.Sampietro et al., Mol Cell 24, 293 (2006)). Equal amounts (1 nM) ofHis-tagged BCL9 and GST-tagged β-catenin bound to glutathione-Sepharose4B beads (GE) were incubated overnight at 4° C. in assay buffer (100 mMNa₂PO₄ [pH7.4], 100 μg/mL bovine serum albumin, 0.01% Triton X-100 and4% DMSO). Complexes of His-tagged BCL9 bound to bead-immobilizedGST-tagged β-catenin were isolated by centrifugation, resuspended in 1mL of assay buffer, and 50 μL of slurry incubated in the presence orabsence of SAH-BCL9_(B) or SAH-BCL9_(MUT) (SAH-BCL9_(B)(R359E)) in 500μl assay buffer for 2 hr at room temperature. Glutathione bead-boundproteins were washed twice by centrifugation, eluted, and resolved bygel electrophoresis. GST-β-catenin was detected by Coomassie bluestaining and the presence of retained His-BCL9 was detected byimmunoblot analysis (anti-His 23655, Cell Signaling) and quantifiedusing ImageJ software (rsbweb.nih.gov/ij). The experiment was repeatedthree times with similar results. The results demonstrated thatSAH-BCL9_(B) could dose-responsively dissociate the complex with an IC₅₀of 135 nM, whereas single point mutagenesis reduced the activity by6-fold. See results FIG. 17B.

Example 23. SAH-BCL9_(B) Inhibits Wnt Transcriptional Activity

To measure the effects of vehicle, SAH-BCL9_(B), and SAH-BCL9_(MUT) onthe expression of Wnt/β-catenin target genes, including VEGF, in Colo320(FIG. 18) and MM1S (FIG. 19) cell lines, quantitative PCR (qRT-PCR)analysis was performed. RNA was extracted with TRIzol Reagent(Invitrogen) according to the manufacturer's protocol. Total RNA (2 μg)was reverse transcribed (SuperScript VILO cDNA synthesis kit,Invitrogen) and qPCR was performed using an Applied Biosynthesis 7500Real-time PCR system. PCR primers were designed as below:

FOXQ 1: (SEQ ID NO: 122) cgcggactttgcactttgaa; (SEQ ID NO: 123)agctttaaggcacgtttgatggag CDK4: (SEQ ID NO: 124) atgttgtccggctgatgga;(SEQ ID NO: 125) caccagggttaccttgatctcc Axin2: (SEQ ID NO: 126)cggaaactgttgacagtggat; (SEQ ID NO: 127) ggtgcaaagacatagccagaa VEGF:(SEQ ID NO: 128) catgaactttctgctgtcttgg; (SEQ ID NO: 129)atgattctgccctcctcctt LGR5: (SEQ ID NO: 130) ctcccaggtctggtgtgttg;(SEQ ID NO: 131) gtgaagacgctgaggttgga CMYC: (SEQ ID NO: 132)tttttcgggtagtggaaaacc; (SEQ ID NO: 133) gcagtagaaatacggctgcac CD44:(SEQ ID NO: 134) tttgcattgcagtcaacagtc; (SEQ ID NO: 135)tgagtccacttggctttctgt CLDN2: (SEQ ID NO: 136) cggtgtggctaagtacaggc;(SEQ ID NO: 137) caaagctcacgatggtggtct LEF-1: (SEQ ID NO: 138)catcccttcctcattccttcaac; (SEQ ID NO: 139) aggcttcctaaaaggtggtgg

Analysis of target genes was conducted in quadruplicate using POWER SYBRGreen Master Mix (Applied Biosystems) as previously described (M. Maniet al., Cancer Res 69, 7577 (2009)). Transcripts levels were normalizedto β-actin expression. These experiments were repeated three times.Treatment with SAH-BCL9_(B), but not vehicle or SAH-BCL9_(MUT),dose-responsively reduced the mRNA levels of VEGF, c-MYC, LGR5, LEF1,and AXIN2 (FIG. 18A and FIG. 19A). Actin, a non-Wnt pathway target gene,was used as a reference in Colo320 cells and showed no change inresponse to SAH-BCL9_(B) treatment (FIG. 18A). LGR5 was reduced inColo320 cells but not in MM1S cells, consistent with the cellularspecificity of Wnt target gene transcription.

To further investigate the specificity of SAH-BCL9_(B) in blocking Wnttranscriptional activity, comparative genome-wide expression analyses ofWnt target genes in the DLD1 colon cancer cell line, for which a Wnttranscription pathway signature has been described (L. G. Van der Flieret al., Gastroenterology 132, 628 (2007)), was performed. RNA fromtriplicate SAH-BCL9_(B)- and vehicle-treated DLD1 samples (10 μM eachfor 12 hours) was isolated for gene expression profiling analyses.Affymetrix Human U133 Plus 2.0 arrays were processed using the functionof the affy Bioconductor package (URL http://www.R-project.org.). Genesets were compiled from Van der Flier et al. and gene set enrichment andstatistical analyses performed using GSEA software(http://www.broad.mit.edu/GSEA) and a two-tailed t-test, respectively.Microarray data has been deposited in the Gene Expression Omnibus(http://www.ncbi.nlm.nih.gov/geo) and comply with MIAME annotationstandards.

The triplicate data sets from SAH-BCL9_(B)- and vehicle-treated DLD1generated using Affymetrix oligonucleotides microarrays were comparedwith published gene expression data from DLD1 cells bearing inducibledominant-negative forms of TCF1 and TCF4 (L. G. Van der Flier et al.,Gastroenterology 132, 628 (2007)). Gene set enrichment analysis (GSEA)revealed a strong and statistically significant correlation between thegenes down-regulated by SAH-BCL9_(B) and the dominant-negative forms ofTCF1 and TCF4 in both adenoma (FIG. 18B, family-wise error (FWER)p-value<0.001; false discovery rate (FDR) q-value<0.001) and carcinoma(FIG. 18C FWER and FDR<0.01), highlighting the specificity ofSAH-BCL9_(B) in blocking Wnt transcriptional activity. Axing, a robustand specific Wnt target gene(3), was among the most down-regulated genesby SAH-BCL9_(B) treatment, in addition to other Wnt targets involved incell metastasis (CD44, CLDN2), cell proliferation (CyclinA2, CDK4), andEMT (FOXQ1) (FIG. 18D and FIG. 18E). These findings were then validatedby qRT-PCR (FIG. 18F). VEGF-A was among the genes downregulated in cellstreated with SAH-BCL9_(B) (FIG. 18F and FIG. 19), linking theβ-catenin/BCL9 complex to tumor-induced angiogenesis.

Example 24. Combination Therapies

To test whether the anti-proliferative effect of SAH-BCL9_(B) couldsynergize with other agents commonly used to treat MM or CRC,combination treatment studies were conducted. Indeed, the cytotoxiceffects of 5-fluorouracil on CRC cells and of doxorubicin on MM cellswere enhanced by SAH-BCL9_(B), but not by vehicle or the mutant peptide.See results in FIG. 21.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, co-pending patent applications,and GenBank numbers) cited throughout this application are herebyexpressly incorporated herein in their entireties by reference. Unlessotherwise defined, all technical and scientific terms used herein areaccorded the meaning commonly known to one with ordinary skill in theart.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

What is claimed is:
 1. A method of treating cancer mediated byBCL9/β-catenin binding in a human subject, the method comprisingadministering to the human subject a therapeutically effective amount ofa structurally constrained peptide of an HD2 domain of BCL9 (BCL9-HD2)comprising at least one hydrocarbon staple, wherein the at least onehydrocarbon staple links the side chain of a first non-natural aminoacid that replaces a first BCL9-HD2 amino acid selected from the groupconsisting of Leu-351, Ser-352, Gln-353, Glu-354, Gln-355, His-358,Arg-359, Arg-361, Ser-362, Leu-363, Thr-365, Leu-366, Ile-369, Gln-370,Met-372, Leu-373, and Phe-374 with the side chain of a secondnon-natural amino acid that replaces a second BCL9-HD2 amino acidselected from the group consisting of Leu-351, Ser-352, Gln-353,Glu-354, Gln-355, Leu-356, Glu-357, His-358, Arg-359, Glu-360, Arg-361,Ser-362, Leu-363, Thr-365, Leu-366, Arg-367, Asp-368, Ile-369, Gln-370,Arg-371, Met-372, Leu-373, and Phe-374, wherein the first and secondnon-natural amino acids are two, three, or six amino acids apart, andwherein the cancer is selected from the group consisting of colorectalcancer, colon cancer, and multiple myeloma.
 2. The method of claim 1,wherein the human subject has been previously identified as in need of acanonical Wnt/β-catenin signaling inhibitor to treat the cancer.
 3. Themethod of claim 1, wherein the human subject is administered with anadditional therapeutic agent, radiation, or chemotherapy.
 4. The methodof claim 3, wherein the additional therapeutic agent is an anti-cancercompound.
 5. The method of claim 3, wherein the structurally constrainedpeptide and the additional therapeutic agent are administeredsimultaneously or sequentially.
 6. The method of claim 1, wherein thestructurally constrained peptide comprises an interacting face comprisedof amino acids that interact with β-catenin, wherein the interactingface comprises BCL9 residues Gln-355, His-358, Arg-359, Ser-362,Leu-363, Leu-366, Ile-369, Gln-370, Leu-373, and Phe-374.
 7. The methodof claim 6, wherein the interacting face represents a single face of anα-helix.
 8. The method of claim 7, wherein the single face of a helixcomprises one, two, three, or four adjacent stacked columns of aminoacids, wherein the stacked columns of amino acids are defined bypositions a, d, and g; positions b and e; or positions c and f; in analpha helix having 3.6 amino acids per turn wherein the amino acids areconsecutively and serially assigned positions a-g; and positions a andd; positions b and e; or positions c and f in a 3₁₀ helix having 3 aminoacids per turn wherein the amino acids are consecutively and seriallyassigned positions a-f; or homologues thereof.
 9. The method of claim 1,wherein the at least one hydrocarbon staple is formed by an olefinmetathesis reaction.
 10. The method of claim 1, wherein the first andsecond non-natural amino acids are selected from the following:


11. The method of claim 1, wherein the structurally constrained peptidecomprises 1 to 2 staples within the BCL9 HD2 peptide.
 12. The method ofclaim 1, wherein the at least one hydrocarbon staple is located at anyof the staple positions in any one of the following BCL9 HD2 stapledpeptides: i, i + 4 single staples: (SEQ ID NO: 8)XSQEXLEHRERSLQTLRDIQRBLF (SEQ ID NO: 9) LXQEQXEHRERSLQTLRDIQRBLF(SEQ ID NO: 10) LSXEQLXHRERSLQTLRDIQRBLF (SEQ ID NO: 11)LSQXQLEXRERSLQTLRDIQRBLF (SEQ ID NO: 12) LSQEXLEHXERSLQTLRDIQRBLF(SEQ ID NO: 13) LSQEQXEHRXRSLQTLRDIQRBLF (SEQ ID NO: 14)LSQEQLXHREXSLQTLRDIQRBLF (SEQ ID NO: 15) LSQEQLEXRERXLQTLRDIQRBLF(SEQ ID NO: 16) LSQEQLEHXERSXQTLRDIQRBLF (SEQ ID NO: 17)LSQEQLEHRXRSLXTLRDIQRBLF (SEQ ID NO: 18) LSQEQLEHREXSLQXLRDIQRBLF(SEQ ID NO: 19) LSQEQLEHRERXLQTXRDIQRBLF (SEQ ID NO: 20)LSQEQLEHRERSXQTLXDIQRBLF (SEQ ID NO: 21) LSQEQLEHRERSLXTLRXIQRBLF(SEQ ID NO: 22) LSQEQLEHRERSLQXLRDXQRBLF (SEQ ID NO: 23)LSQEQLEHRERSLQTXRDIXRBLF (SEQ ID NO: 24) LSQEQLEHRERSLQTLXDIQXBLF(SEQ ID NO: 25) LSQEQLEHRERSLQTLRXIQRXLF (SEQ ID NO: 26)LSQEQLEHRERSLQTLRDXQRBXF (SEQ ID NO: 27) LSQEQLEHRERSLQTLRDIXRBLXi, i + 7 staples: (SEQ ID NO: 28) XSQEQLEXRERSLQTLRDIQRBLF(SEQ ID NO: 29) LXQEQLEHXERSLQTLRDIQRBLF (SEQ ID NO: 30)LSXEQLEHRXRSLQTLRDIQRBLF (SEQ ID NO: 31) LSQXQLEHREXSLQTLRDIQRBLF(SEQ ID NO: 32) LSQEXLEHRERXLQTLRDIQRBLF (SEQ ID NO: 33)LSQEQXEHRERSXQTLRDIQRBLF (SEQ ID NO: 34) LSQEQLXHRERSLXTLRDIQRBLF(SEQ ID NO: 35) LSQEQLEXRERSLQXLRDIQRBLF (SEQ ID NO: 36)LSQEQLEHXERSLQTXRDIQRBLF (SEQ ID NO: 37) LSQEQLEHRXRSLQTLXDIQRBLF(SEQ ID NO: 38) LSQEQLEHREXSLQTLRXIQRBLF (SEQ ID NO: 39)LSQEQLEHRERXLQTLRDXQRBLF (SEQ ID NO: 40) LSQEQLEHRERSXQTLRDIXRBLF(SEQ ID NO: 41) LSQEQLEHRERSLXTLRDIQXBLF (SEQ ID NO: 42)LSQEQLEHRERSLQXLRDIQRXLF (SEQ ID NO: 43) LSQEQLEHRERSLQTXRDIQRBXF(SEQ ID NO: 44) LSQEQLEHRERSLQTLXDIQRBLX i, i + 3 single staples:(SEQ ID NO: 45) XSQXQLEHRERSLQTLRDIQRBLF (SEQ ID NO: 46)LXQEXLEHRERSLQTLRDIQRBLF (SEQ ID NO: 47) LSXEQXEHRERSLQTLRDIQRBLF(SEQ ID NO: 48) LSQEXLEXRERSLQTLRDIQRBLF (SEQ ID NO: 49)LSQEQXEHXERSLQTLRDIQRBLF (SEQ ID NO: 50) LSQEQLXHRXRSLQTLRDIQRBLF(SEQ ID NO: 51) LSQEQLEXREXSLQTLRDIQRBLF (SEQ ID NO: 52)LSQEQLEHXERXLQTLRDIQRBLF (SEQ ID NO: 53) LSQEQLEHRXRSXQTLRDIQRBLF(SEQ ID NO: 54) LSQEQLEHREXSLXTLRDIQRBLF (SEQ ID NO: 55)LSQEQLEHRERXLQXLRDIQRBLF (SEQ ID NO: 56) LSQEQLEHRERSXQTXRDIQRBLF(SEQ ID NO: 57) LSQEQLEHRERSLXTLXDIQRBLF (SEQ ID NO: 58)LSQEQLEHRERSLQXLRXIQRBLF (SEQ ID NO: 59) LSQEQLEHRERSLQTXRDXQRBLF(SEQ ID NO: 60) LSQEQLEHRERSLQTLXDIXRBLF (SEQ ID NO: 61)LSQEQLEHRERSLQTLRXIQXBLF (SEQ ID NO: 62) LSQEQLEHRERSLQTLRDXQRXLF(SEQ ID NO: 63) LSQEQLEHRERSLQTLRDIXRBXF (SEQ ID NO: 64)LSQEQLEHRERSLQTLRDIQXBLX.


13. The method of claim 1, wherein the structurally constrained peptideof BCL9-HD2 comprises two hydrocarbon staples and the two hydrocarbonstaples are located at any of the positions within any one of thefollowing BCL9 HD2 stapled peptides: i, i + 3 double staples:(SEQ ID NO: 65) XSQXQLEHRERSLQTLRDIQXBLX (SEQ ID NO: 66)XSQXQLEHRERSLQTLRDIXRBXF (SEQ ID NO: 67) XSQXQLEHRERSLQTLRDXQRBXFi, i + 4 double staples: (SEQ ID NO: 68) XSQEXLEHRERSLQTLRDIXRBLX(SEQ ID NO: 69) XSQEXLEHRERSLQTLRDXQRBXF (SEQ ID NO: 70)XSQEXLEHRERSLQTLXDIQRXLF i, i + 7 double staples: (SEQ ID NO: 71)XSQEQLEXRERSLQTLXDIQRBLX (SEQ ID NO: 72) XSQEQLEXRERSLQTXRDIQRBXF(SEQ ID NO: 73) XSQEQLEXRERSLQXLRDIQRXLF.


14. The method of claim 1, wherein the at least one hydrocarbon stapleis located at any of the positions within any one of the following BCL9HD2 stapled peptides: Mixed i, i + 4; i, i + 3; and i, i + 7 double staples: (SEQ ID NO: 74) XSQEXLEHRERSLQTLXDIQRBLX (SEQ ID NO: 75)XSQEXLEHRERSLQTXRDIQRBXF (SEQ ID NO: 76) XSQEXLEHRERSLQXLRDIQRXLF(SEQ ID NO: 77) XSQEXLEHRERSLQTLRDIQXBLX (SEQ ID NO: 78)XSQEXLEHRERSLQTLRDIXRBXF (SEQ ID NO: 79) XSQEXLEHRERSLQTLRDXQRXLF(SEQ ID NO: 80) XSQEQLEXRERSLQTLRDIXRBLX (SEQ ID NO: 81)XSQEQLEXRERSLQTLRDXQRBXF (SEQ ID NO: 82) XSQEQLEXRERSLQTLRXIQRXLF(SEQ ID NO: 83) XSQEQLEXRERSLQTLRDIQXBLX (SEQ ID NO: 84)XSQEQLEXRERSLQTLRDIXRBXF (SEQ ID NO: 85) XSQEQLEXRERSLQTLRDXQRXLF(SEQ ID NO: 86) XSQXQLEHRERSLQTLRDIXRBLX (SEQ ID NO: 87)XSQXQLEHRERSLQTLRDXQRBXF (SEQ ID NO: 88) XSQXQLEHRERSLQTLRXIQRXLF(SEQ ID NO: 89) XSQXQLEHRERSLQTLXDIQRBLX (SEQ ID NO: 90)XSQXQLEHRERSLQTXRDIQRBXF (SEQ ID NO: 91) XSQXQLEHRERSLQXLRDIQRXLFSequential i, i + 4 staples: (SEQ ID NO: 92) XSQEXLEHXERSLQTLRDIQRBLF(SEQ ID NO: 93) LXQEQXEHRXRSLQTLRDIQRBLF (SEQ ID NO: 94)LSXEQLXHREXSLQTLRDIQRBLF (SEQ ID NO: 95) LSQXQLEXRERXLQTLRDIQRBLF(SEQ ID NO: 96) LSQEXLEHXERSXQTLRDIQRBLF (SEQ ID NO: 97)LSQEQXEHRXRSLXTLRDIQRBLF (SEQ ID NO: 98) LSQEQLXHREXSLQXLRDIQRBLF(SEQ ID NO: 99) LSQEQLEXRERXLQTXRDIQRBLF (SEQ ID NO: 100)LSQEQLEHXERSXQTLXDIQRBLF (SEQ ID NO: 101) LSQEQLEHRXRSLXTLRXIQRBLF(SEQ ID NO: 102) LSQEQLEHREXSLQXLRDXQRBLF (SEQ ID NO: 103)LSQEQLEHRERXLQTXRDIXRBLF (SEQ ID NO: 104) LSQEQLEHRERSXQTLXDIQXBLF(SEQ ID NO: 105) LSQEQLEHRERSLXTLRXIQRXLF (SEQ ID NO: 106)LSQEQLEHRERSLQXLRDXQRBXF (SEQ ID NO: 107) LSQEQLEHRERSLQTXRDIXRBLXSequential i, i + 3 staples: (SEQ ID NO: 108) XSQXQLXHRERSLQTLRDIQRBLFSequential i, i + 7 staples: (SEQ ID NO: 109) XSQEQLEXRERSLQXLRDIQRBLFMixed sequential staples: (SEQ ID NO: 110) XSQXQLEXRERSLQTLRDIQRBLF(SEQ ID NO: 111) XSQXQLEHREXSLQTLRDIQRBLF (SEQ ID NO: 112)XSQEXLEXRERSLQTLRDIQRBLF (SEQ ID NO: 113) XSQEXLEHRERXLQTLRDIQRBLF(SEQ ID NO: 114) XSQEQLEXREXSLQTLRDIQRBLF (SEQ ID NO: 115)XSQEQLEXRERXLQTLRDIQRBLF.


15. The method of claim 1, wherein the structurally constrained peptidecomprises an amino acid sequence selected from the group consisting of:SAH-BCL9_(A): (SEQ ID NO: 3) LSQEQLEHRERSLQTLRXIQRXLF; SAH-BCL9_(B):(SEQ ID NO: 4) LSQEQLEHRERSLXTLRXIQRBLF; and SAH-BCL9_(C):(SEQ ID NO: 5) LSQEQLEHREXSLQXLRDIQRBLF.


16. The method of claim 15, wherein the structurally constrained peptidecomprises the amino acid sequence of SEQ ID NO:
 3. 17. The method ofclaim 15, wherein the structurally constrained peptide comprises theamino acid sequence of SEQ ID NO: 4.